**Myxothiazol A** (Fig. 21 A, top) is a natural compound obtained from the predatory bacterium, *Myxococcus fulvus*.⁴⁹⁷,⁴⁹⁸ It is the best known representative of the thiazole-based bacterial toxins that possess a β-methoxyacrylamide or β-methoxyacrylate (MOA) toxophores.⁴⁷⁴,⁴⁹⁹,⁵⁰⁰ Myxothiazol is considered a classic P<sub>m</sub> type Q<sub>o</sub> inhibitor - its binding does not involve the immobilisation of the IPS-HD in the *b*-position. Based on the structure of the bovine Cytbc₁ co-crystallized with myxothiazol ⁷⁷, it has been proposed that the inhibitor molecule is bound to the Q<sub>o</sub> pocket, mostly through interactions of the dithiazole moiety with aromatic residues in the binding site and by a characteristic hydrogen bond formed between the amide oxygen atom of myxothiazol and the backbone amide of the conserved Glu in the PEWY motif. Moreover, the amide nitrogen of myxothiazol can form a hydrogen bond with the conserved Tyr of the PEWY motif. It was also suggested that the C18 methyl group of myxothiazol can serve as an unusual hydrogen bond donor to the sulfur atom of BtMet124. It is worth mentioning, that myxothiazol-related compounds that demonstrate inhibitory properties towards the Q<sub>o</sub> site are more abundant. In the case of *M. fulvus* toxins, myxothiazol Z, which comprises a β-methoxyacrylate moiety, has also been shown to exhibit antifungal properties *in vivo*.⁵⁰¹ This group also includes the **melithiazols** (Fig. 21 A, middle) isolated from *Melittangium lichenicola* and several other strains ⁵⁰² and **cystothiazoles** (Fig. 21 A, bottom) from *Cystobacter fuscus* ⁵⁰³, which were shown to exhibit antifungal properties *in vivo*.⁵⁰²,⁵⁰³ In the enzymatic assays with bovine SMP's, melithiazol A was shown to exert a similar spectroscopic effect on redox cofactors to that of myxothiazol.⁵⁰² Therefore, it can be expected that these inhibitors have a similar mode of binding to the Q<sub>o</sub> site.

Another group of myxobacterial compounds, **cyrmenins** (Fig. 21 B), were obtained from *Cystobacter armeniaca* and *Archangium gephyra*. Cyrmenins are distinct from myxothiazol-related compounds, and comprise a MOA moiety substituted at the α-position.⁵⁰⁴ **Cyrmenin B₁** has been shown to inhibit Cytbc₁, but the binding mode was not investigated in details.⁵⁰⁵ The presence of the MOA toxophore suggests that cyrmenin targets the Q₀ site as well.
<figure><img src="image_25.png" /><figcaption>**Figure 21.** MOA-comprising myxobacterial toxins. A) Structural formulae of natural $Q_0$ site inhibitors with dithiazole moieties: myxothiazol A (top) comprising β-methoxyacrylamide toxophore and two other, comprising the β-methoxyacrylate toxophore, melithiazol A (middle) and cystothiazole A (bottom). B) Structural formula of cyrmenin $B_1$. Pale-red, dotted frames indicate the toxophore moiety of each compound.</figcaption></figure>

**Strobilurins** (Fig. 22) are currently recognized as a large group of synthetic $P_m$ class $Cytbc_1$ inhibitors with various structures and toxophore moieties. However, such generalization can be confusing, and in fact they were all designed using natural fungal poliketydes as a template. These compounds were isolated from several species of large, wood-decaying Basidiomycetes, initially named strobilurins, eg. **strobilurin A** (also known as mucidin <sup>506</sup>), (Fig. 22 A left) from *Strobiluris tenacellus* <sup>507</sup> and **oudemansin A** (Fig. 22 A, right) from *Oudemansiella mucida* <sup>508</sup>. Later an Ascomycete, *Bolinea lutea* was also reported to produce strobilurins and their derivatives <sup>509</sup>. Natural strobilurins possess the β-methoxyacrylate (MOA) toxophore<sup>499,510</sup> (Fig. 22, **1** in red frames), which targets the heme $b_L$ proximal part of the $Q_0$ site<sup>511,512</sup>. In contrast to myxothiazol-like inhibitors, the β-methoxyacrylate moiety of strobilurins is substituted with a tail at the α-position, not the β-position. Due to this difference, their distinct origin, and their separate paths of biosynthesis<sup>513</sup>, myxothiazol and other myxobacterial compounds should not be referred to as “strobilurins”. The presence of MOA in both groups of compounds should be rather taken as a biochemical example of evolutionary convergence. Natural strobilurins exhibit a low photostability<sup>510</sup>. Therefore, various synthetic compounds (Fig. 22 B) with differently substituted MOA toxophores (Fig. 22, **1** in red frames) have been created, such as **MOA-stilbene**<sup>57</sup>, **azoxystrobin**<sup>77</sup> or **picoxystrobin**<sup>510,514</sup>. Several deviations
from classic MOA toxophore were developed (Fig. 22 B, **2-8** in red frames), such as oximino-acetate
(2) e.g. **kresoxim-methyl** ⁵¹⁰,⁵¹⁴ and **trifloxystrobin** ⁵¹⁰,⁵¹⁴, oximino-acetamide (3) e.g.
**dimoxystrobin** ⁵¹⁰, methyl N-methoxycarbamate (4) e.g. **pyraclostrobin**⁵¹⁴,⁵¹⁵, triazolinone ⁵¹⁶ (5)
e.g. **JZZ**¹¹⁷, tetrazolinone (6) eg. **metyltetraprole**⁵¹⁷, dihydro-dioxazine (7) eg. **fluoxastrobin**⁵¹⁰ and
the methyl-carbamate (8) e.g. **pyribencarb**.⁵¹⁸ Such diversity of synthetic strobilurin analogues is
mostly a result of “patent wars” between agrochemical companies, driven by a large demand from
the global fungicidal market.⁵¹⁴ Despite apparent differences in structure, man-made strobilurins are
expected to share the same binding mode to the Q₀ site as the classic MOA inhibitors. This has been
partially confirmed by the results of X-ray crystallographic studies on Cytbc₁ with bound MOA-
stilbene⁷⁷,⁸³, azoxystrobin⁷⁷, trifloxystrobin⁴⁷⁴, iodinated derivative of kresoxim-methyl⁴⁷⁴, and JZZ
(PDB ID:3L73)⁵¹⁹, showing that the toxophores of these inhibitors superimpose within the binding
site, with formation of a hydrogen bond to the backbone amide of the conserved PEWY motif Glu
residue. Among the synthetic strobilurin-like inhibitors mentioned here, pyribencarb has been
suggested as a novel, “benzylcarbamate” type of Q₀ inhibitor.⁵¹⁸ Indeed, it has been shown to possess
a higher selectivity than other MOA analogues. However, neither its methyl-carbamate toxophore nor
binding mode are as unique as has been claimed because methyl-carbamate moiety is present in
previously described natural crocacins, and also in macrolides isolated from sponges, such as
**neopeltolide** (Fig. 23 A, upper) from unidentified *Daedalopelta* sp.⁵²⁰,⁵²¹ and **leucascandrolide A**
(Fig. 23 A, lower) from *Leucascandra caveolata*.⁵²² Enzymatic assays confirmed that these
compounds also exhibit inhibitory properties towards Cytbc₁.⁵²³ Moreover, the recent molecular
modeling docking studies revealed that pyribencarb, crocacin and neopeltolide all bind to the Q₀ site
through their methyl-carbamate moieties, in a way similar to the MOA moiety of azoxystrobin,
forming a hydrogen bond to the backbone amide group of BtGlu271.⁵²⁴
<figure><img src="image_26.png" /><figcaption>**Figure 22.** Structural formulae of selected natural (A) and synthetic (B) strobilurins. *Pale-red*, *dotted* frames indicate the toxophore moiety of each compound. Numbers within frames indicate the type of toxophore (explained in text).</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>**Figure 23.** Structural formulae of (A) macrolides isolated from sea sponges, with their methyl-carbamate toxophores marked with frames, and (B) pyrimorph. *Pale-red*, *dotted* frames indicate the toxophore moiety of each compound.</figcaption></figure>
**Pyrimorph** (Fig. 23 B) is a synthetic morpholine derivative, already in use as a fungicide against pathogens of plants.<sup>525-527</sup> It has been shown to exhibit inhibitory activity towards multiple targets, including cellulose synthase A<sup>526</sup> and Cytbc<sub>1</sub>.<sup>527</sup> Results of enzymatic assays indicate that pyrimorph acts as a non-competitive inhibitor of the Q<sub>o</sub> site. Moreover, complementary molecular modeling studies showed that pyrimorph docks in the central cavity between Cytbc<sub>1</sub> monomers, at the entrance of the Q<sub>o</sub> site, and thus blocks access of the substrate. The binding mode of pyrimorph has no resemblance to any other Q<sub>o</sub> site inhibitor. It was found that it does not form hydrogen bonds within the Q<sub>o</sub> site, but instead it is held in place by hydrophobic and van der Waals interactions.<sup>527</sup> Thus, pyrimorph should be considered a unique inhibitor type, distinct from MOA-related compounds.

<figure><img src="image_28.png" /><figcaption>Figure 24. Structural formulae of dual-mode inhibitors of Cytbc<sub>1</sub>. A) 1-hydroxyquinolin-4-ones. B) Tautomeric states of 1-hydroxyquinolin-4-one. C) Ametoctradin. D) Quinolones. E) Ascochlorin.</figcaption></figure>

*Dual-mode inhibitors*. Some inhibitors are capable of binding to both Q<sub>i</sub> and Q<sub>o</sub> catalytic sites, therefore they should be considered as a third, dual-mode class of Cytbc<sub>1</sub> inhibitors. They are often quinone analogues or compounds semi-isosteric with quinones, and therefore able to mimic the natural substrate within both sites.

The 2-alkyl derivatives of 1-hydroxyquinoline-4-one, such as **HQNO**, **NQNO** or **HDQ** (Fig. 24 A) are usually considered Q<sub>i</sub> site inhibitors, with a mechanism of action similar to that of antimycin A.<sup>111,438,528</sup> Some early reports suggested that HQNO and NQNO bound to Cytbc<sub>1</sub> can be displaced by both antimycin A and UHDBT.<sup>529,530</sup> This effect was explained as a possible result of allosteric interactions between the two binding sites.<sup>530</sup> However, more recent crystallographic analysis has shown that NQNO is indeed capable of binding to both Q<sub>i</sub> and Q<sub>o</sub> sites. It has been proposed that in
solution, NQNO most likely exists as a mixture of two tautomers (Fig. 24 B), which is likely a
determinant factor in this dual mode of binding. At the Qᵢ site, the NQNO tautomer with its hydroxyl
group protonated (Fig. 24 B, left) can form a hydrogen bond to ⁶⁹Ser205, while the form with N-oxide
protonated (Fig. 24 B, right) can hydrogen bond with ⁶⁹Asp228. In the structural model obtained in
these studies, the ⁶⁹His201 imidazole ring was not in the correct orientation for hydrogen bonding. At
the Q₀ site, the N-oxide protonated form of NQNO can form two hydrogen bonds. The N-oxide
oxygen forms a water-mediated hydrogen bond with ⁶⁹Glu271 of the PEWY motif, and the 4-carbonyl
oxygen forms a hydrogen bond with the His (⁶⁹His161) ligand of 2Fe2S cluster, immobilizing the
ISP-HD in *b*-position.⁷⁹ However, there is no data to confirm the dual-mode binding for other 1-
hydroxyquinoline-4-ones. Recent studies suggest that HQNO induces changes in the environment of
the Q₀ site, but not due to binding in the site.¹¹¹

**ELQ-400** (Fig. 24 D, upper) is a 4(1H)-quinolone, one among a large number of derivatives of the antimalarial drug, **endochin** (Fig. 24 D, lower) ⁴⁴¹,⁵³¹. Most of ELQ's (endochin-like quinolones) are expected to inhibit the Qᵢ site of apicomplexan *Cytbc*₁.⁴⁴¹,⁵³¹,⁵³² Thus, it was proposed that they be used in combination with the Q₀ site-targeting atovaquone.⁵³² However, it has recently been suggested that the binding mode of ELQ-400 is more complex, with the inhibitor targeting both active sites ⁵³³. This proposal was strongly supported by mutational analysis and molecular modeling studies. At the Qᵢ site, ELQ-400 was predicted to bind in a similar manner to the substrate or pyridone inhibitors (mentioned earlier). At the Q₀ site, it was predicted to bind similarly to atovaquone or stigmatellin, with a hydrogen bond formed by the NH group of the quinolone to the His ligand of 2Fe2S cluster.⁵³³

**Ametoctradin** (Fig. 24 C) is a triazolopyrimidine derivative, acting as a highly selective inhibitor against the *Cytbc*₁ of oomycetes.⁴⁶⁹,⁵³⁴,⁵³⁵ It has been shown to be active against mammalian (porcine)⁴⁶⁸, but inactive towards yeast⁵³⁵ *Cytbc*₁. Ametoctradin is commonly used in agriculture and available under trade name Initium®.⁵³⁴ No cross-resistance of ametoctradin with MOA-like inhibitors has been observed, so it is speculated to bind in the Q₀ site in a manner similar to stigmatellin.⁵³⁵ However, the molecular modeling docking studies have shown that although ametoctradin binds in roughly the same part of Q₀ pocket as stigmatellin, and forms a hydrogen bond to the carboxylate side chain of the conserved PEWY motif Glu residue, the distance to the 2Fe2S-ligating His residue is too far to expect formation of a strong hydrogen bond.⁴⁶⁸ In addition there is no experimental indication that ISP-HD is immobilized by ametoctradin in the *b*-position, as in the presence of stigmatellin. More recent studies suggest that ametoctradin is a dual-mode inhibitor, targeting both the Qᵢ and Q₀ site.⁵³⁶ This model seems to be supported by the observation that ametoctradin is inactive towards the wild-type strain of *Saccharomyces cerevisiae* and active towards
a mutant with an altered Qᵢ site. Based on this finding it was suggested that, like NQNO, ametoctradin
can bind to both active sites.

**Ascochlorin** (Fig. 24 E) is an isoprenoid compound, which was independently isolated from four distinct fungal strains (*Ascochyta viciae* 537,538, *Cylindrocladium ilicicola* 454, *Fusarium* sp. LL-Z 1272 539 and *Acremonium luzulae* 540) and described by four different research groups at about the same time. Because of this remarkable coincidence, it can be found in the literature and databases under three different names: ascochlorin, LL-Z 1272γ and ilicicolin D 541. In the recent comprehensive biochemical, spectroscopic and crystallographic studies, ascochlorin has been shown to bind to both Qᵢ and Q₀ sites of bacterial, yeast, mammalian and avian *Cytbc*₁ 542. At the Qᵢ site the position of the aromatic ring of bound ascochlorin overlaps with the position of the bound ubiquinone aromatic ring. Moreover, at the Qᵢ site, it has been proposed that ascochlorin forms a halide bond between the Cl atom and the conserved His (the proton donor in Q reduction) and a hydrogen bond between its hydroxyl group in the para position to Cl and the conserved Asp (the second proton donor in Q reduction). At the Q₀ site, ascochlorin occupies a position similar to stigmatellin. A hydrogen bond, is formed, possibly from both the hydroxyl group and the C=O group to the His ligand of 2Fe2S cluster, immobilizing ISP-HD at the *b*-position. On the other side of the aromatic ring, the Cl atom is suggested to form a halide bond with Glu in the conserved PEWY motif. However, as it has been pointed out that a mixture of conformations may exist, this remains uncertain. The dual mode of action can be explained in terms of ascochlorin and related compounds being in general, ubiquinone analogs, as they can mimic natural substrates also in other ubiquinol oxidoreductases.⁵⁴³

The inhibitors of *Cytbc*₁ already comprise numerous groups of compounds, both natural and synthetic, with new ones being discovered or developed almost every year.⁵²⁴,⁵⁴⁴ Many were omitted in this section, as little is known about their mechanism of action, and any attempt to deduce or model it would go beyond the scope of this review. Some recent discoveries in the field are rather unexpected, for example the recent findings with **karrikinolide** (Fig. 25, upper), a naturally occurring byproduct from the combustion of plant material, which earlier had been shown to stimulate plant seed germination.⁵⁴⁵ Karrikinolide itself exhibits almost negligible inhibitory properties towards *Cytbc*₁. However, some of its synthetic derivatives, such a 4-*n*-butylphenyl-substituted variant (Fig. 25, lower), have been shown to inhibit porcine *Cytbc*₁ with efficiency comparable to commercially available inhibitors ⁵⁴⁶.
<figure><img src="image_29.png" /><figcaption>Figure 25. Structural formulae of karrikinolide (upper) and its 4-*n*-butylphenyl derivative (lower panel).</figcaption></figure>

**5.2 Cytochrome *b*<sub>6</sub>*f*-specific inhibitors**

Several inhibitors have been employed to characterize the functional and structural features
of the Cytb<sub>6f</sub>. These compounds act on both the Q<sub>p</sub> and the Q<sub>n</sub> sites similarly to the situation described
above for the Cytbc<sub>1</sub>.

*Inhibitors of the* Q<sub>p</sub> *site*. Typical inhibitors of this site are: DNP-INT, stigmatellin and DBMIB.

DNP-INT (2,4- dinitrophenylether of 2-iodo-4-nitro-thymol) (Fig. 26) belongs to the family of dinitrophenyl ethers <sup>547</sup>, i.e., compounds that compete with plastoquinol for its oxidising pocket without having redox properties. These compounds do not act as electron donors or redox mediators, unlike DBMIB (see below). DNP-INT inhibits photosynthetic electron flow *in vitro*, where its activity can be circumvented by addition of TMPD (N-tetramethyl-p-phenylenediamine), an artificial electron carrier able to mediate e- flow from PQH<sub>2</sub> to the Cytf (Trebst 1980). DNP-INT is also active *in vivo* as demonstrated by the work of Delosme and colleagues in *Chlorella sorokiniana* <sup>548</sup>. In this organism, DNP-INT lowers the rate of Cytb<sub>6</sub> oxidation and of PC reduction under single turnover flash conditions. Furthermore, DNP-INT diminishes the amplitude of the slow phase of the Electro-chromic Band Shift (ECS), a modification of the absorption spectrum of intra-membrane pigments in response to the transmembrane electrical field (ca. 3x10<sup>5</sup> V/cm) an expression of the quantum-mechanical Stark Effect, following the establishment of the proton motive force across the thylakoids membrane, <sup>549</sup>. The ECS slow phase (phase B) has been proposed to be a consequence of the transfer of an electron across the low potential chain of the cytochrome complex<sup>550</sup>. A study by Fitzpatrick and colleagues challenged the efficiency of DNT-INT as an inhibitor of photosynthetic electron flow. A possible explanation for this finding is that DNP-INT would be a poor inhibitor in
continuous light, while efficiently blocking Cytb<sub>6f</sub> under single turnover illumination, as previously reported<sup>551</sup>.

<figure><img src="image_30.png" /><figcaption>Figure 26. Chemical structure of DNP-INT inhibitor</figcaption></figure>

**Stigmatellin** (Fig. 18A) is a potent antibiotic obtained from the myxobacterium *Stigmatella aurantiaca*, which inhibits both the Cytbc<sub>1</sub> and Cytb<sub>6f</sub>.<sup>552</sup> This compound binds to the PQH<sub>2</sub> oxidising site, as shown by crystallographic studies in cyanobacteria and green algae<sup>41,42</sup>, although not in the same position in the two organisms. Stigmatellin interacts with the 2Fe2S, raising its midpoint potential (in isolated Cytb<sub>6f</sub>) from 320 to about 460 mV<sup>553</sup>. This increase in potential is accompanied by changes in the EPR spectrum of the 2Fe2S cluster. *In vivo*, its action mechanism is similar to that DNP-INT.<sup>551</sup> Under single turnover flash illumination, stigmatellin reduces, in parallel, the rate of Cytf and of Cytb<sub>6</sub> reduction in a concentration-dependent manner.

In Cytbc₁, stigmatellin has been extensively used to probe conformational changes related to the moment of ISP-HD from a proximal site, i. e. close to the quinol, to a distal site, close to Cytc (reviewed in Berry et al. ¹). This movement is required to facilitate electron flow in the high potential chain, and to promote electron bifurcation, as predicted by the Q cycle mechanism ¹. In Cytb₆f, the transition between these two positions has not been resolved by X-ray crystallographically in the same clear cut manner as in Cytbc₁. Nonetheless, electron crystallography ⁵⁵⁴ has underlined movements of the ISP-HD in the Cytb₆f, via the comparison of projection maps of thin three-dimensional crystals of Cytb₆f prepared with or without stigmatellin. 2D crystallography has also suggested possible conformational changes in the transmembrane region of the complex, upon stigmatellin binding.

Unlike other inhibitors of Cytb<sub>6f</sub>, stigmatellin binds not only to the luminal but also to the stromal side of the Cytb<sub>6f</sub><sup>66,173</sup>. The Q<sub>n</sub> site has a lower affinity for stigmatellin than does the p-side lumenal site. In the Q<sub>n</sub> site stigmatellin faces the quinone exchange cavity, being in interaction with Arg 207 of the Cytb<sub>6</sub> and the supplementary heme c<sub>n</sub>. (see also section 3.6.2).
**DBMIB** (2,5-dibromothymoquinone) (Fig. 18 C) is probably the most representative of a class of inhibitors based on halogenated or hydroxylated lipophilic benzoquinones⁵⁴⁷. DBMIB has been extensively used as a specific inhibitor of PQH₂ oxidation. Early results by Haehnel indicate that it does not affect PQ reduction by PSII, while blocking its reoxidation by PSI⁵⁵⁵. The number of DBMIB molecules required for inhibition is one per 300 chlorophylls, i.e. one per electron-transport chain. This would argue in favour of a 1 to 1 binding stoichiometry with *Cytb*6f. However, studies of the consequences of DBMIB on the EPR spectrum of the 2Fe2S by Roberts and Kramer led to the conclusion that two molecules of DBMIB bind to each monomer of the spinach *Cytb*6f, both in the isolated form and in intact thylakoid membranes⁵⁵⁶. Binding likely occurs in two sites: a high affinity one, where DBMIB binding would cause small shifts in the EPR spectrum of the 2Fe2S similar to those induced by stigmatellin, and a low-affinity site, where binding was induced by super-stoichiometric amounts of the inhibitor. DBMIB binding is accompanied by the appearance of a new EPR signal (g = 1.94). The binding of two molecules of DBMIB to the Qp/Qo pocket would be consistent with the so-called “double-occupancy” models proposed in the case of *Cytbc*₁ (see paragraph **Erreur ! Source du renvoi introuvable.**) However, crystallographic data unambiguously showed that only one molecule of DBMIB can bind to the PQH₂ oxidation site.⁶⁷ To reconcile these findings, Roberts and coworkers, repeated the EPR spectra with oriented *Cytb*6f¹¹⁰, and found that DBMIB affects the orientation of the ISP-HD within *Cytb*6f depending on the stoichiometry of the inhibitor at the Qp/Qo site. With a single DBMIB, the EPR signatures of the ISP-HD are consistent with this protein being in the proximal position (close to the Qp/Qo pocket), similar to the orientation observed in the X-ray crystal structure of the *Cytb*6f in the presence of DBMIB⁶⁷. With ≥ 2 equiv. of DBMIB bound, the ISP-HD would be in a position resembling the one observed in the *Chlamydomonas reinhardtii*, *Cytb*6f in the presence of tridecylstigmatellin (TDS) ⁴¹, suggesting that the low-affinity DBMIB site is at the distal niche. This conclusion is consistent with earlier EPR measurements of isolated *Cytb*6f poisoned with DBMIB ⁸⁴, and can explain the peculiar mechanism of inhibition of PQH₂ oxidation by this compound observed *in vitro* and *in vivo*.

Early studies by Rich and colleagues ⁵⁵⁷ showed that reduced DBMIB (DBMIBH₂) does not block the *Cytb*6f activity during its first turnover. Thus, DBMIB would bind the complex in its reduced form but only become inhibitory upon its oxidation. The inhibitory complex would thus involve a semiquinone or a quinone form of DBMIB. This hypothesis was subsequently confirmed in vivo ⁵⁵¹. The authors found that DNP-INT affects equally the low- and high-potential chain of *Cytb*6f decreasing the rates of both *Cytf* reduction and *Cytb*₆ turnover. Conversely, DBMIB inhibits only the rate of *Cytf* reduction while reducing, at the same time, the amplitude of *Cytb*₆ redox signals. The
accessibility of DNP-INT to the Qp site was unaffected by preillumination, while that of DBMIB was greatly enhanced, even after a single turnover. Overall, the authors propose that oxidation of DBMIBH₂ by a single turnover flash would trigger the same conformational changes of the ISP-HD that occurs during oxidation of PQH₂ in unpoisoned Cytb6f. This would allow transferring one electron to the high-potential chain, and generating at the same time the inhibitory DBMIB species ⁵⁵¹. Slow reduction of DBMIB inside the Qp pocket (see Rich et al. ⁵⁵⁷) would regenerate DBMIBH₂, which would diffuse out of the quinol binding pocket, unlashing the inhibition. A relatively recent study by Sarewicz/Bujnowicz et. al. revealed that the EPR spectrum of 2Fe2S in DBMIB-supplemented spinach Cytb6f is frequency-dependent which is a strong indication that DBMIB semiquinone at the Qp site is spin-spin coupled to the reduced 2Fe2S ⁴⁸².

Besides being extensively used to characterize electron flow in the photosynthetic chain, DBMIB has also been largely employed to study cell biological responses in photosynthetic organisms. Combining DBMIB with the PSII specific inhibitor DCMU allows the PQ(H₂) pool to be either reduced (DBMIB) or oxidised (DCMU) in the light. Based on this approach, it was proposed that the redox state of the PQ(H₂) pool regulates the transcription of a subset of chloroplast genes to adjust the relative stoichiometric amounts of the two reaction centres (PSII and PSI) (see, e.g. reviews by ⁵⁵⁸⁻⁵⁶⁰). These findings have led to the concept of redox mediated plastid retrograde signalling that orchestrates the expression of genes by the plastid and nuclear genomes, a concept that is still discussed (review in Pfannscheidt 2010 and Chan et al., 2016 ⁵⁶¹,⁵⁶²).

**Ions.** Divalent cations also inhibit the catalytic cycle of the Cytb6f in concentration dependent manner. Inhibition was first reported for Cu²⁺¹¹² and then for Zn²⁺¹⁰⁸. Both metals were proposed to hamper the movement of the ISP-HD. Based on EPR data, it was proposed that binding of the two ions in proximity of His143 in the ISP would induce the inhibition. Later crystallographic studies with Cd²⁺poisoned Cytb6f¹⁷³ provided a structural confirmation of this hypothesis, showing the existence of two Cd²⁺binding sites, the strongest one being mediated by Cd-H143 interactions.

*Inhibitors of the Qn site.* Typical inhibitors of this site are: N(H)QNO, MOA stilbene and, to a lower extent, stigmatellin.

**NQNO** (2-n-nonyl-4-hydroxy-quinoline N-oxide) was first investigated by Jones and Whitmarsh ⁵⁶³ *in vitro* and by Joliot and Joliot ⁵⁶⁴ in *Chlorella sorokiniana* cells. Jones and Whitmarsh ⁵⁶⁵ used single turnover and steady state absorption spectroscopy to show that NQNO binds to the stromal quinone site, thereby inhibiting the oxidation of the Cytb6 heme, and slowing down electron
transfer between the two b-hemes. Based on the effect of NQNO on the ECS kinetics, the authors concluded that: i) the slow phase of the ECS under single turnover flash (phase b, see above) is due to electron transfer between the two *b* hemes, followed by a reaction associated with PQ reduction; ii) the two quinone binding sites would be separated by 70% of the dielectrically weighted distance across the membrane.

Joliot and Joliot further explored the relationship between this inhibitor, redox changes of Cytb₆ and changes in the kinetics and amplitude of phase b of the ECS. Based on the consequences of NQNO poisoning of phase b, the authors proposed that reduction of the quinone occurring at the stromal site (Qn) would be coupled to protonation, implying that this site is connected to the outer face of the thylakoid by a proton channel. Recent crystallographic studies have provided a molecular interpretation for these findings, showing that at least two residues (Asp 20 and Arg 207) form a H⁺ wire connecting the stroma to the quinone binding pocket <sup>66</sup>.

NQNO binds in close proximity to the heme c_n, acting as a direct axial ligand of the heme (Yamashita et al., 2007). This interaction explains why NQNO binding to the Cytb6f leads to a strong modification of the properties of heme c_n<sup>191</sup>. This heme titrates as a one-electron Nernst curve with a E_m value of +100 mV in unpoisoned complexes. Its reduced minus oxidized spectrum displays a broad absorbance increase peaking at approximately 425 nm. Upon binding of NQNO two heme c_n titrations waves are visible: one with an E_m value similar to that observed in non-inhibited enzyme and the other with a midpoint shift by about -225 mV <sup>191</sup>. Moreover, the Soret spectrum of the heme is shifted by 1 nm to longer wavelengths upon NQNO binding <sup>173</sup>

**MOA stilbene** (Fig. 22, 1 in *red frames*) This inhibitor was first studied by Rich and colleagues <sup>566</sup>, who showed that at variance with Cytbc<sub>1</sub>, MOA-stibene does not affect quinol oxidation but instead quinone reduction. Its binding to Cytb<sub>6f</sub> induces a red-shift of the Soret and visible absorbance bands of the *b* hemes, enhancing their ‘oxidant-induced reduction’ and slowing down their subsequent dark reoxidation at the same time. Its effect is therefore similar to that of NQNO.

Later, Kramer and Crofts <sup>218</sup> confirmed these results, showing that MOA-stilbene increases the reduction of the high potential form of Cytb<sub>6</sub> by 45% ca, and reduced the amplitude of the ECS signal by about 25%, again in agreement with results obtained with NQNO.

**Stigmatellin** (Fig. 18 A). As mentioned above, this inhibitor also binds to the Qn site with very similar features as NQNO. However, due to its higher affinity for the Qp site, this inhibitor cannot be used to test the mechanism of electron and proton transfer to PQ.
## 6 Thermodynamic background of cytochrome bc catalysis

This section covers the thermodynamic principles behind operation of Cyt-bc, using the reactions characteristic for Cytbc₁. If the Cytb₆f were to operate according to a “modified Q cycle”, as described for Cytbc₁, then the equations below would be equally applicable to both complexes, simply by exchanging the parameters for PC/Cytc₆ and PQ(H₂) with those of Cytc and UQ(H₂) respectively. However, as described in detail in section 3.6.5, significant doubts remain about the operation of a Q cycle at Cytb₆f, with the possibility that additional electrons may be added to the Qn side from soluble components. In this case, the stoichiometry of the equations below will clearly be altered for the Cytb₆f

In aqueous solutions, the quinone-dependent enzymatic reaction of Cytbc₁ can be summarized as ¹,⁵⁶⁷:

$$2 \text{Cytc}^{3+} + \text{QH}_2 = 2 \text{Cytc}^{2+} + \text{Q} + 2 \text{H}^+ \quad (\text{eq. 10})$$

where Cytc³⁺ and Cytc²⁺ stand for ferri- and ferro-Cytc, respectively.

In this case, the change in ΔG associated with reaction of electron transfer from quinone molecules to Cytc molecules after mixing UQH₂ derivatives with Cytc can be estimated using the relationship ⁵⁶⁸:

$$\Delta G = - nF \Delta E_h \quad (\text{eq. 11})$$

where ΔEh is the difference in the actual redox potential between the couples of the donor (Eh(D) of UQ/UQH₂) and acceptor (Eh(A) of Cytc³⁺/Cytc²⁺) and n is the number of electrons in the reaction (here n = 2) and F is the Faraday constant.

$$\Delta E_h = E_{h(A)} - E_{h(D)} \quad (\text{eq. 12})$$

The corresponding Eh(A) and Eh(D) can be calculated using the Nernst equation which takes into account the redox midpoint potentials at pH 7 (Eₘ₇Q and Eₘ₇Cyt) and actual concentrations of reduced and oxidized form of UQ/UQH₂ and Cytc³⁺/Cytc²⁺, the temperature (T) and the gas constant (R).⁵⁶⁸.

$$E_{h7(A)} = E_{m7}^{Cyt} + RT/nF \ln([\text{Cytc}^{3+}]/[\text{Cytc}^{2+}]) \quad (\text{eq. 13})$$

$$E_{h7(D)} = E_{m7}^{Q} + RT/nF \ln([\text{UQ}]/[\text{UQH}_2]) \quad (\text{eq. 14})$$

During a typical measurement of Cytbc₁ turnover rate, the substrates are added to the solutions in total concentrations that should exceed Michaelis-Menten constants, while proportions of oxidized to reduced substrates are approximately 0.05 and 0.95 for [UQ]/[UQH₂] and [Cytc³⁺]/[Cytc²⁺], respectively ⁵⁶⁹.

Taking +260 mV as the Eₘ at the physiological pH of 7 (Eₘ₇) of Cytc³⁺/Cytc²⁺ ⁵⁷⁰, +70 mV as the Eₘ₇ of Q/QH₂ ⁴⁸⁸ and the number of electrons n = 2, one may calculate the energy released during the oxidation of 1 mol of QH₂ and reduction of 2 mols of Cytc:

$$\Delta G = - nF \Delta E_h = -66 \text{ kJ/mol.} \quad (\text{eq. 15})$$
For comparison, the ΔG of oxidation of 1 mol of NADH in complex I and reduction of O₂ in complex IV in the mitochondrial respiratory chain is approximately -224 kJ/mol ¹⁶².

However, for the enzyme operating in living cells, eq. 11 is too simple by far and ignores an important fact that Cytc reduction by Cytbc₁ is associated with the coupling of two oppositely directed redox reactions taking place at the Qo/Qp and Qi/Qn catalytic sites. Moreover, these reactions are coupled to the release of protons to the p side, and uptake of protons from the n side of membrane. Therefore, the simple eq. 10 should be split into two equations in order to account for this spatial separation of the two different quinone redox reactions in Cytbc₁:

$$QH_2^{(p)} + Cytc^{3+} + Q^{(n)} = Q^{(p)} + Cytc^{2+} + SQ^{-(n)} + 2H^{+(p)} \quad (\text{eq. 16})$$

$$QH_2^{(p)} + Cytc^{3+} + SQ^{-(n)} + 2H^{+(n)} = Q^{(p)} + Cytc^{2+} + QH_2^{-(n)} + 2H^{+(p)} \quad (\text{eq. 17})$$

Superscripts (p) and (n) denotes that the respective reaction takes place at the Qo/Qp and Qi/Qn site, respectively.

These reactions lead to proton transfer across the membrane in which Cytbc is embedded, i.e. 4 protons are released (from Qo/Qp site) to the positive side (p side), while 2 protons are taken (at the Qi/Qn site) from the n side of the membrane. In this way the proton transfer catalyzed by the enzyme contributes to an increase of the transmembrane electrochemical potential gradient (Δp) which is used to drive the energetically uphill reaction of ADP to ATP conversion in cells ¹⁶². However, it is worth mentioning here that the protons are transferred across the membrane by coupling deprotonation of QH₂ at p side with protonation of Q at n side, according to Mitchelian redox loop mechanisms. This is different from other complexes such as CcO for example which catalyzes active pumping of protons through specific proton channels.

Proton motive force is a value expressed as the sum of two components: difference in proton concentration between two sides of membrane (ΔpH) and the electric potential difference (Δψ) between the two sides of membrane. However, for simplicity Δp is commonly converted to equivalent differences in electric potential expressed in millivolts.

In mitochondria, ΔpH is usually small and thus Δp is largely dominated by Δψ, which is on the order of 150 – 200 mV¹⁶², depending on the actual state of mitochondria (ADP/ATP ratio, oxygen tension and the activity of decoupling proteins). The architecture of Cytbc imposes an additional energetic barrier to electron transfer from the Qo/Qp site through heme bL/bp and bH/bn to the Qi/Qn site.
site as the electrons must be transferred against $\Delta\psi$, when the enzyme is embedded in the coupled membrane$^{161,571-573}$. Therefore equation 15 must take into account this additional energetic barrier:

$$ \Delta G = -nF(\Delta E_h - \Delta\psi) \quad (\text{eq. 18}) $$

Under conditions in which the Q(H₂) pool and Cytc/PC pool are half reduced, the driving force is significantly lowered. As a result, increasing $\Delta\psi$ influences the ET process hence increasing the probability of reduction of heme $b_L/b_p$ at the expense of heme $b_H/b_n$. Under extreme conditions, $\Delta\psi$ can be high enough to impair the QH₂ oxidation at the Q₀/Qₚ site leading to an increase in superoxide generation during the catalysis$^{572}$.

# 7 Mechanistic insights into the catalytic Q₀ / Qₚ site

## 7.1 Overview of the structure of the Q₀ site

Cytb of Cytbc₁ is built of 8 transmembrane helices arranged in two helical bundles with the first bundle (helices A to E) incorporating both hemes $b$ ($b_L$ and $b_H$). The second bundle (helices F to H) is located in the vicinity of heme $b_L$ and together with the large helix-connecting loops at the intermembrane (periplasmic) side of the membrane, forms the Q₀ site binding pocket$^{20,21,79,132}$. This pocket is highly hydrophobic and contains many highly conserved residues. The major loop regions that participate in formation of the Q₀ site include the two helices cd1 and cd2 of the CD loop and the ef helix of the EF loop. The helices cd1 and cd2 cap the pocket from the intermembrane (periplasmic) space where Cytb interacts with the ISP-HD. Transient binding of the ISP-HD to Cytb brings the 2Fe2S cluster into proximity with the substrate occupying the Q₀ site pocket, which means that in this pocket the substrate can be flanked by two redox active cofactors: the 2Fe2S and heme $b_L$. This corresponds to the state when the two cofactors required for electron bifurcation are present close enough to the substrate bound at the Q₀ site for fast electron transfer to occur (the exact distance is not known but approximated from the crystal position of the Q₀ site inhibitors, see below). Due to the large-scale oscillatory movement of the ISP-HD between the Cytb and Cytc₁, there are states of the Q₀ site with 2Fe2S is not present at the site and therefore unable to interact with the bound substrate (precluding the bifurcation reaction). The movement itself is an inherent part of the catalytic cycle as it connects the Q₀ site with heme c of Cytc₁: transient binding of the ISP-HD away from Cytb to Cytc₁ brings the 2Fe2S and heme c₁ close enough for fast electron transfer between these two cofactors.
## 7.2 Quinone binding to the Q₀ site

Binding of the QH₂ molecule to the Q₀ site is an obvious requirement for initiating the catalytic QH₂ oxidation. Despite many years of study the way in which Q or QH₂ are bound to this site is still not fully understood. A major part of what is known about these interactions comes from measuring site-directed mutants of bacterial *Cytbc*₁, usually of conserved residues at the Q₀ site, in spectroscopic studies, mainly EPR spectroscopy and X-ray crystallography. The latter method has always been considered as the most promising tool for detection of specific chemical interactions between substrates and the protein. However, soon after solving the first structures of *Cytbc*₁, it became clear that in the case of this protein (as in some other quinone-binding proteins) the crystallography failed to show natural substrate bound at the Q₀ site and neither Q nor QH₂ has been resolved within this site²⁰. It has been proposed that this failure is due to the Q molecules at the Q₀ site being in a very dynamic state. However, a simple loss of the substrate during the isolation and crystallization of the protein also cannot be ruled out, in particular in the light of spectroscopic observations that samples of isolated *Cytbc*₁ tend to have an empty Q₀ site⁵⁹,¹²⁷,²⁶⁰.

In the absence of structures containing natural substrate at the Q₀ site, structures obtained with many different site-specific inhibitors have served as a starting point for the construction of models for Q binding to Q₀. Several of these proposals, including the most recent ones, considered specific interactions derived after substituting the chromane ring of stigmatellin with the natural quinone ring⁹¹,⁹⁵,⁹⁶,⁵⁷⁴,⁵⁷⁵.

In studying the binding of QH₂ and Q to the Q₀ site, EPR spectroscopy of the reduced 2Fe2S has been one of the most widely used methods. It has benefited from the observation that, for *Cytbc*₁, the continuous wave EPR (CW-EPR) spectra of 2Fe2S is highly sensitive to variation in the type of molecule occupying the Q₀ site and its redox state, providing a convenient means to monitor the status of the site in the native membranes. This approach seems to also be applicable for detection of PQ or PQH₂ bound to the Qₚ site of *Cytb*ₕ.⁵⁷⁶ From the very beginning it became apparent that CW-EPR spectra of 2Fe2S could not be simulated by modeling a single EPR component for the 2Fe2S¹⁵⁹ nor by using a statistical theory of *g* strain that has previously worked for other iron-sulfur clusters⁵⁷⁷.

The sensitivity of the CW-EPR spectra of the reduced 2Fe2S to the occupant of the Q₀ site is best documented for bacterial *Cytbc*₁. It was observed that the shape of the spectrum differed depending whether *Cytbc*₁ was embedded in native membranes or isolated and solubilized in detergent micelles¹²⁷. It was also observed that the shape of the spectrum in the membranes was sensitive to the total quinone content in the membrane¹⁰⁹,²⁶⁰,⁵⁷⁸,⁵⁷⁹ as well as being a convenient tool for discriminating between Q or QH₂ occupancy of the Q₀ site⁵⁸⁰.
In bacterial chromatophores, the most characteristic spectral feature of 2Fe2S is the $g_x$ transition which equals 1.800 when UQ bound at the Q₀ site interacts with the cluster⁵⁷⁹. Among all the transitions of the 2Fe2S spectrum, the $g_x$ component appears the most influenced by the occupant of the Q₀ site. When the site contains UQH₂, $g_x$ broadens significantly and shifts to approximately 1.777¹⁰⁹,⁵⁷⁸,⁵⁷⁹. Other transitions ($g_z$, $g_y$) are also relatively narrow when UQ is bound to the Q₀ site suggesting a relative small g-strain of the cluster⁹⁰,¹¹¹,¹¹³,⁵⁷⁹,⁵⁸¹. These transitions also broaden, although less significantly comparing to $g_x$, when UQ exchanges with UQH₂.

Careful analysis of the $g_x$ transition has established that measurements of relative proportions of $g_x$ at 1.800 vs $g_x$ at 1.777 signal can be used to monitor the redox state of the Q(H₂) pool in the membrane. Most probably the $g_x = 1.800$ transition results from interaction of the 2Fe2S environment with the carbonyl group of UQ, while the $g_x = 1.777$ results from interaction with the hydroxyl group of UQH₂⁵⁷⁸. However, this sensitivity of the 2Fe2S spectrum to UQ or UQH₂ is completely abolished when low-molecular-weight alcohols, such as methanol, ethanol, glycerol (a common cryoprotectant), are present⁵⁸². This most likely results from interaction of the hydroxyl group with the $RhH156$ ligand of 2Fe2S ($BtH161$). It follows that measurements of the redox state of the Q(H₂) pool by CW-EPR spectra of 2Fe2S (which must be performed at low temperatures) must avoid cryoprotective agents as they usually contain hydroxyl groups⁵⁸³. On the other hand, the absolute necessity of using cryportectants in low-temperature pulse EPR measurements compromises sensitivity of the 2Fe2S spectra to UQ and UQH₂. Otherwise formation of ice may cause aggregation and denaturation and may significantly increase the efficiency of paramagnetic relaxation distorting the results, which are dependent on spin-lattice relaxation rates.

For the empty Q₀ site, the $g_x$ transition is very broad and its g value approaches 1.765⁵⁸⁰. A similar shape and g value for the $g_x$ transition is detected when the Q₀ site is occupied by the inhibitor myxothiazol or MOA-stilbene⁵⁷⁸.

Interestingly, the experiments on partial extraction of UQ from purple bacterial membranes (chromatophores) revealed that when the ratio of the number of Q molecules per Q₀ sites is between 2:1 and 1:1, the $g_x$ value changes to approximately 1.78 and its linewidth lies in-between $g_x = 1.800$ (for UQ) and 1.777 (for UQH₂)⁵⁸⁴. Furthermore, this 1.78 transition could not be reproduced by a linear combination of “UQ $g_x$ 1.800” and “empty-site $g_x$ 1.765” lines. To explain these observations Ding et al, proposed that there are two niches, strong and weak quinone binding at Q₀: Q₀s and Q₀w, with dissociation constants ($K_d$) of approximately ~0.05 and ~1 mM, respectively²⁶⁰. Taking these $K_d$ values and assuming that, under normal conditions, the effective concentration of UQ in the membrane is ~30 mM²⁶⁰, it was concluded that the Q₀ site should be occupied by two molecules of UQ at the same time. This lead the authors to formulate a “double-occupancy” model, according to
which the Q₀ site can accommodate 2 UQH₂ molecules simultaneously²⁶⁰,⁵⁷⁸,⁵⁸⁰. Further support for the double occupancy model has come from determination of the stoichiometry of UQH₂ binding to Cytbc₁ by NMR-based analysis of inhibitor displacements. With this approach, two molecules of UQ(H₂) were shown to bind specifically to the Q₀ site.⁵⁸⁵ This observation was incorporated into a model that aimed to explain the mechanism of bifurcation. It was proposed that UQH₂ and UQ binds at the same time in the Q₀ site and undergo a comproportionation reaction according to eq. 7, resulting in formation of two SQ molecules, each donating one electron to the c- or b- chain⁵⁸⁵,⁵⁸⁶. However, this concept has not been verified with other, more direct studies.

In the opinion of the authors of this review, the double-occupancy model remains problematic and it was previously the subject of debate⁹¹. When measuring the redox state of the Q(H₂) pool in samples poised at ambient redox potentials (*E*<sub>h</sub>) equal to or lower than the *E*<sub>m</sub> of the UQ/UQH₂ couple, the Q₀ site would be occupied by UQ at the Q₀s niche with an increasing population of UQH₂ at the Q₀w niche as *E*<sub>h</sub> shifts negatively. If this happened during redox titration of the membranous UQ(H₂) pool, as monitored by EPR measurements of the 2Fe2S spectrum, the *g*<sub>x</sub> would likely be different from the observed experimentally linear combination of *g*<sub>x</sub> = 1.800 (corresponding to UQ<sup>Qos</sup>UQ<sup>Qow</sup>) and 1.777 (corresponding to UQH<sub>2</sub><sup>Qos</sup>UQH<sub>2</sub><sup>Qow</sup>) transitions. Such a titration should also reveal a separate component corresponding to the unique UQ<sup>Qos</sup>UQH<sub>2</sub><sup>Qow</sup> state or respective molecules after comproportionation. Alternatively, the presence of two non-equivalent sites for UQ(H₂) at the Q₀ site would very likely lead to a non-Nernstian shape of the redox titration curve for the UQ/UQH₂ couple, detected in the *g*<sub>x</sub> transition of 2Fe2S. However, none of these observations can be made, and the titration curve in such experiments closely follows the Nernst equation with n = 2¹⁰⁹,⁵⁷⁹. In addition, the *E*<sub>m</sub> values of UQ/UQH₂ obtained from EPR measurements of the 2Fe2S spectra remain consistent with spectrophotometric measurements of the redox midpoint potential of this couple⁵⁸⁷. This suggests that UQ and UQH₂ at the Q₀ site are in dynamic equilibrium with UQ and UQH₂ of the membranous pool. Going further, one may conclude that if changes in EPR spectra follow the redox state of the UQ(H₂) pool, UQ is not favorably bound to the site over UQH₂ and *vice versa*.

The first step of the QH₂ oxidation at the Q₀ site involves binding of QH₂ and association of the ISP-HD with an oxidized 2Fe2S to the Q₀ site. Thus, the initial step of the reaction is formation of the enzyme-substrate (ES) complex, which depends on equilibrium constants describing the ratio between the population of Q₀ sites with QH₂ bound and those which are empty (K<sub>QH2-Qo</sub>). Additionally, the association constant of ISP-HD containing oxidized 2Fe2S with the Q₀ (K<sub>ISPox</sub>) site is also a crucial parameter defining the probability of ES complex formation. While direct designation of K<sub>QH2-Qo</sub> and K<sub>ISPox</sub> is not experimentally possible, the respective values for Cytbc₁ were approximated by Crofts et al.²³ who considered the following states:
$$K_{QH_2-Q_0} = [Q_0-QH_2] / [Q_0-\text{empty}] \sim 10 \quad (\text{eq. 19})$$

$$K_{\text{ISPox}} = [Q_0-QH_2-\text{ISP-HD}_{Q_0}] / [Q_0-\text{ISP-HD}_{\text{remote}}] \sim 10 \quad (\text{eq. 20})$$

where: [Q₀-QH₂] and [Q₀-empty] are the concentrations of QH₂ at the Q₀ site and empty Q₀ site, respectively, at the time when ISP-HD containing oxidized 2Fe2S cluster is in position close to the Q₀ site. [Q₀-QH₂-ISP-HDQ₀] and [Q₀-ISP-HDremote] denote relative concentrations of ISP-HD containing oxidized 2Fe2S at position close to the Q₀ site with bound QH₂ and remote from the site, respectively.

From eq. 19 it can be concluded that both QH₂ and oxidized 2Fe2S have a tendency to form the ES complex to initiate the first step of electronic bifurcation. Unfortunately, a direct observation of the interaction of QH₂ with oxidized 2Fe2S has not yet been made, as such a state can be generated only under non-equilibrium conditions, which would make the 2Fe2S diamagnetic and therefore not detectable by EPR spectroscopy. The EPR spectroscopy performed with cryoreduction of the 2Fe2S cluster in oriented membranes is also not informative. Although it can detect the position of ISP-HD with oxidized 2Fe2S, the Q₀ site contains only UQ, not QH₂¹⁰⁰.

## 7.3 Crucial amino acid residues involved in the binding and catalysis of QH₂ / PQH₂ oxidation at the Q₀/Qₚ site

This paragraph focuses on some of the crucial amino acid residues of the Q₀ site that are thought to be involved in Q(H₂) binding, and of the Qₚ site, involved in PQ(H₂) binding. At a general level, understanding how individual structural elements contribute to Q(H₂) or PQ(H₂) binding is challenging because of the difficulty in defining specific and universal Q(H₂) binding motifs in proteins⁵⁸⁸ and the possibly related observation that many protein-Q(H₂) interactions involve atoms of the main polypeptide chain.

Nevertheless, extensive mutational studies targeting residues of the Q₀ site has identified those that affect catalysis, although some of these mutations were found to primarily affect the stability and/or assembly of the whole protein complex. It is of note that, despite a large number of mutations that have already been tested in the region of the Q₀ site, relatively few were able to completely abolish enzymatic activity, implying significant robustness and tolerance for structural change of both Q(H₂) binding and Q₀ site catalysis.

Current models of Q and QH₂ binding to the Q₀ site are derived from crystallographic structures of Cytbc₁ with bound inhibitors replacing the natural substrate (structures with the substrate have not been solved yet). The models consider both Pₘ and Pₚ inhibitors, i.e. inhibitors exerting
different effects on the motion of ISP-HD as well as those occupying different niches of the Q₀ pocket,
(see section 5 for details). The latter difference is visualized by a comparison of the structures shown
in Fig. 27, which compare binding of myxothiazol (representative of P_m group) at a position closer to
heme b_L and further from ISP-HD with binding of stigmatellin (representative of P_f inhibitors) –at a
position closer to ISP-HD and further from heme b_L.

7.3.1 **RhH156 (BtH161) in iron-sulfur protein and the RhE295 (BtE271) in**
**cytochrome b**

<figure><img src="image_31.png" /><figcaption>**Figure 27.** Comparison of structure of the Q₀ site from *Bos taurus* with bound myxothiazol (A) or stigmatellin (B). Prepared on basis of 1SQP<sup>77</sup> and 1PP9<sup>123</sup>, respectively. Amino acids of ISP are not shown in A, since myxothiazol induce c-position of ISP-HD. The imidazole ring of the 2Fe2S ligand – histidine 161, is shown in B as a structure forming an H bond to stigmatellin.</figcaption></figure>

The two most prominent, and thus most frequently discussed, candidates for residues that directly participate in binding substrate are histidine BtH161 (RhH156) - one of the ligands of the 2Fe2S and glutamate Bt271 (RhE295) from conserved PEWY motif of Cytb. A binding mode for substrate involving these two residues is thought to be equivalent to the configuration of stigmatellin in the crystal structure. Stigmatellin forms two strong H bonds in the Qo site (Fig. 27 B): one between the >C=O group of the inhibitor and N<sub>t</sub> atom of BtH161 of ISP-HD and second between -OH group and carboxylic group of BtE271 residue<sup>123</sup>. Interestingly, myxothiazol crystalised in the Qo site does not form specific hydrogen bonds to either of these groups. A comparison of the two structures reveals variable positions for the BtE271 side chain: it rotates further toward the chromane ring of stigmatellin in order to create a hydrogen bond with the -OH group at the C8 atom of the ring. It is highly unlikely that Q or QH<sub>2</sub> molecules at the Qo site simultaneously form H bonds to BtH161 and BtE271 as
stigmatellin does. Therefore it was proposed that during the oxidation QH₂ undergoes a rapid movement between the two niches within the Q₀ site filled by stigmatellin and myxothiazol<sup>23,575,589,590</sup>. According to this mechanism, QH₂ binds to the Q₀ via <sup>Bt</sup>H161 (<sup>Rh</sup>H156) and the first electron transfer to the oxidized 2Fe2S takes place. The first proton is released and then the neutral SQH moves to form a hydrogen bond to <sup>Bt</sup>E271. The Glu residue promotes deprotonation of SQH, which then donates an electron to heme b<sub>L</sub>. After formation of Q, it shifts back to the position that allows interaction with <sup>Bt</sup>H161 and then it can be replaced with another QH₂. This mechanism has not been proven yet and it is a still a matter of debate<sup>259</sup>. While the SQ moving between the two niches should be trappable, the experiments generally fail to detect such an intermediate of reaction (see section 7.6). Importantly, it remains an open question as to whether stigmatellin is actually a good model for Q(H₂) binding. The spatial separation of the two -OH groups differs between the QH₂ and the chromane ring of stigmatellin, with consequences for the capacity to form H bonds. Comparing structures crystallized with different inhibitors, it seems that binding of UHDBT<sup>77</sup> or atovaquone<sup>71</sup> at the Q₀ site might more closely reflect binding of QH₂ as the structures of these inhibitors are more similar to natural quinones, (menaquinone and sulfolobus- or caldariellaquinone for example<sup>591</sup>), than the chromane ring of stigmatellin.

The idea that <sup>Bt</sup>H161 might participate in binding of Q and QH₂ to the Q₀ was proposed after ENDOR measurements revealed that the 2Fe2S is coordinated by 2 Cys and 2 His side chains <sup>592</sup> which was later confirmed by crystallographic structures <sup>593</sup>. Electrochemical studies revealed that the E<sub>m</sub> value of 2Fe2S is pH dependent, and therefore dependency requires two different pK values<sup>594</sup>. It was estimated that the pK<sub>a</sub> for oxidized 2Fe2S is ~7.5 and increases to more than 10 upon reduction of the 2Fe2S. This suggests that the protonation of <sup>Bt</sup>H161 is tightly coupled to the redox state of the 2Fe2S. Such an increase in pK means that the energy of hydrogen bond formation between an occupant of the Q₀ and <sup>Bt</sup>H161 is larger when 2Fe2S is reduced compared to when the 2Fe2S is oxidized. Stabilization of the ISP-HD by the reduced 2Fe2S through interaction with Q(H₂) occupying the Q₀ site is also inferred from EPR measurements. Typically, the EPR spectra of the reduced 2Fe2S in chromatophore membranes reflect close interactions with the Q(H₂), revealing that the reduced state of 2Fe2S promotes ISP-HD occupancy of a position close to the Q₀ site (see section 3.4). On the other hand, experiments in which Cytbc₁ containing oxidized 2Fe2S was frozen before the cluster was reduced for detection by EPR showed that the ISP-HD with oxidized cluster tends to occupy a position remote from the Q₀ site.<sup>100</sup>

The 2Fe2S coordinating <sup>Bt</sup>His161 is well established as a crucial amino acid residue for the activity of all Cyt-bc. Its role in formation of initial ES complex is supported by many biochemical
and computational studies. However, all mutational studies aimed at replacing this residue result in
loss of the whole ISP subunit<sup>138</sup>, making definitive proof elusive.

The majority of the models describing formation of the initial ES complex assume that $^{B}$His161 must be deprotonated before binding the QH$_{2}$, whereas Q can bind to the Q$_{o}$ site when it is already protonated$^{595-597}$. In some models, this His residue has therefore been proposed as the first proton acceptor during the oxidation of QH$_{2}$. However, MD simulations indicate that QH$_{2}$ can also form a stable H bond to the His, even when it has already been protonated.$^{95,96}$

The role and significance of the conserved PEWY domain Glu residue in binding substrate and catalytic reaction are less understood<sup>23,118,598</sup>. In several models <sup>Bt</sup>E271 is considered to directly accept the second proton from QH<sub>2</sub> upon its oxidation. However, mutational studies of this residue in the bacterial enzyme revealed only a modest impact on turnover rate and no significant changes in the estimated binding affinity of QH<sub>2</sub> to the Q<sub>o</sub> site<sup>579</sup>. On the other hand, mutation of the same residue in yeast (<sup>Sc</sup>E272Q or <sup>Sc</sup>E272D) induced stigmatellin resistance<sup>598</sup>. These two observations suggest that <sup>Bt</sup>E271 may not be directly involved in either binding of Q or QH<sub>2</sub> to the Q<sub>o</sub> site nor be critically required for proton uptake from QH<sub>2</sub> during the bifurcation reaction. Rather, it may be residue that increases stability of the water network at the Q<sub>o</sub> site, which is necessary to create an appropriate environment for efficient and fast proton removal from the Q<sub>o</sub> site. Clearly, further studies are needed to clarify the role of this residue in binding and/or proton events taking place at the site.

Interesting evolutionary aspects of the PEWY motif are beyond the scope of this review but interested readers will find a discussion on this subject in appropriate reference<sup>118</sup>.

### 7.3.2 Cytb:$^{Rh}$Y147 ($^{Bt}$Y131) and Cytb:$^{Rh}$Y302 ($^{Bt}$Y278)

The Y147 residue is conserved among all Cytb$^{c1}$ complexes. Its importance in the activity of the Qo site was postulated before crystallographic structures were available⁵⁹⁹. Mutations of this residue to Phe, Val, Ser or Ala have a dramatic effect on the activity of bacterial Cytb$^{c1}$⁵⁹⁹. This significant impairment of catalysis is not associated with a change in the redox potentials of heme bL nor 2Fe2S. MD studies imply that Y147 can rotate to form a hydrogen bond between the -OH group at position 4 of UQH₂ and the -COO⁻ group of $^{Rh}$E295⁹⁶, suggesting that this residue might play an important role in formation of the ES complex and the process of proton uptake during the bifurcation reaction. Quantum mechanics calculations performed on MD optimized structures, indicate that when UQH₂ interacts with protonated ISP:$^{Rh}$H156, proton transfer from the hydroxyl group of $^{Rh}$Y147 to the carboxyl group of $^{Rh}$E295 takes place, creating a negative $^{Rh}$Y147 and neutral $^{Rh}$E295. At the same time, the deprotonated hydroxyl of $^{Rh}$Y147 forms a hydrogen bond to the O1 atom of the hydroxyl
group of UQH₂. When similar calculations were performed using the structure in which the hydroxyl O4 atom of UQH₂ forms a hydrogen bond to the already protonated <sup>Rh</sup>H156 of ISP-HD, and the H atom of the ubiquinol -OH group interacts with water molecules, this proton transfer from <sup>Rh</sup>Y147 to <sup>Rh</sup>E295 was not observed, despite the fact that <sup>Rh</sup>Y147 could still perform a relay function between UQH₂ and <sup>Rh</sup>E295<sup>96</sup>. Other MD simulations on the binding of UQH₂ to the Q₀ site did not show involvement of <sup>Rh</sup>Y147 hydroxyl in interaction with UQH₂. Instead, a hydroxyl group at the C1 atom of UQH₂ formed a hydrogen bond with a network with water molecules<sup>95</sup>.

Although a possible role of <sup>Rh</sup>Y147 and <sup>Rh</sup>E295 in binding and proton uptake from UQH₂ during the bifurcation reaction was proposed on the basis of biochemical and mutational studies<sup>590,599</sup>, the concept of H bond network formation between UQH₂-<sup>Rh</sup>Y147-<sup>Rh</sup>E295 clearly needs further investigation.

An important role for Cytb:<sup>Rh</sup>Y302 (<sup>Sc</sup>Y279) in the binding and oxidation of QH₂ by the Q₀ site emerged from several studies on bacterial and mitochondrial Cytbc₁. Mutating this residue to Leu, Gly or Glu in R. sphaeroides Cytbc₁, was found to decrease catalytic rates by a factor between 3 - fold to 50 - fold<sup>589,600</sup> and to increase ROS generation<sup>601</sup>. These effects were also reported for <sup>Sc</sup>Y279C, a mutation associated with mitochondria-related diseases<sup>602</sup>. The gₓ transition of the EPR spectra of 2Fe2S in this mutant suggested that the Q₀ site was “partially empty”<sup>600</sup>. Furthermore, the spectrum of 2Fe2S with bound stigmatellin differed from the equivalent spectrum for typical wild-type enzymes, indicating much stronger binding of the chromane than the benzoquinone ring to the Q₀ site of Cytbc₁. Analysis of the structure of yeast Cytbc₁ with the inhibitor HHDBT bound suggested that <sup>Sc</sup>Y279 might contribute to the binding of UQH₂ by creation of a H bond to the =O atom of the main chain of <sup>Sc</sup>C180 in ISP-HD<sup>70</sup>. Such a hydrogen bond is expected to stabilize UQH₂ binding with <sup>Sc</sup>H181 (<sup>Rh</sup>H156 and <sup>Bt</sup>H161) of the ISP-HD.

In *Rhodobacter capsulatus*, the <sup>Rh</sup>Y302C mutation exerted a deleterious effect on stability of 2Fe2S due to disruption of a disulfide bridge in ISP-HD and resulting cross-linking between the Cytb subunit and ISP-HD (<sup>Rh</sup>Y302C-<sup>Rh</sup>C155). This made the 2Fe2S more sensitive to oxidative damage<sup>601,603</sup>.

In *Plasmodium falciparum* PfY268S (equivalent *Sc*Y279S and 7*Rh*Y302S) was identified as a mutation responsible for resistance of the parasite to the anti-malarial drug, atovaquone<sup>604</sup>. This potent inhibitor of the Q<sub>o</sub> site was found to completely block activity of the yeast Cytbc<sub>1</sub> (IC<sub>50</sub> = 5 nM)<sup>605</sup>. Atovaquone arrests the ISP-HD at the Q<sub>o</sub> site in a similar way to stigmatellin, despite the fact that unlike stigmatellin it does not form a direct H-bond with the conserved PEWY sequence Glu residue<sup>71,606</sup>. The crystal structure revealed that *Sc*Y279 forms hydrogen bonds to Cys180 in the ISP but it also interacts weakly with ionized hydroxyl group at C3. The loss of this Tyr residue in
*Plasmodium* is not lethal to the parasite, but significantly slows its Cytbc₁ activity, thus slowing the parasite growth rate⁶⁰⁴.

### 7.3.3 Other residues involved in substrate binding to the Q₀ site

Besides the amino acid side chains discussed above, it is expected that the binding of substrate to the Q₀ site also engages other residues in the site, including atoms of the main polypeptide chain of Cytb and ISP. Indeed, MD simulations investigating possible structures of bound UQH₂ and UQ at the Q₀ site identified the =O atoms of RhI292 (Cytb) and RhC155 (ISP) as potential residues participating in binding of UQH₂ and UQ through water molecules.

Another residue important for binding of the substrate to the Q₀ site is conserved Gly RhG158 (ScG143 and BtG142) in the WGA sequence²⁶⁰,⁵⁶⁹,⁶⁰⁷. Mutagenesis studies have revealed that its replacement with residues that have bulkier side chains, particularly tryptophan, prevents binding of either UQ(H₂) or inhibitors such as stigmatellin. This effect is most probably due to a steric hindrance at the Q₀ site, preventing catalytic activity of the enzyme.

It should be noted that the isoprenoid chain of UQ also constitutes an important part of the Q binding interface. Indeed, several hydrophobic residues of Cytb interact with this tail. In *R. capsulatus* they are: F144, F166, F298, F337, I162, I169, V333, M336, L165, V161, F298, G141, M140, L201, L178⁹⁵.

### 7.3.4 Specific residues involved in PQ/PQH₂ binding to the Qₚ site of Cytochrome *b*₆f

Studies to characterize residues of Cytb₆f involved in PQ(H₂) binding at the Qₚ site are more limited than in the case of the Cytbc₁ counterpart. These studies have been mainly performed in *Chlamydomonas*, and have revealed strong similarities between the Qₚ and Q₀ sites of Cytb₆f and Cytbc₁, a concept that was later confirmed by crystallographic studies.

The first characterized mutant (FUD50) bears a 36 base pair duplication in the chloroplast petB gene, leading to a 12 amino acid duplication in the CD loop of Cytb₆. This modification decreases the affinity of the Qₚ site for PQ(H₂)⁶⁰⁸, likely by increasing the size of the Qₚ site itself. Subsequent studies have also focused on this region of the Qₚ site, revealing the essential role of the PEWY sequence in Q(H₂) binding and proton release. Later on, this concept that has been rationalized based on the structures of the Cytb₆f in this alga and cyanobacteria⁴¹,⁴²,⁶⁶.

Additional mutants have been made in *Chlamydomonas* to study the role of the ISP flexible hinge in catalytic turnover of the complex. Six Gly residues in the flexible hinge critical for domain
movement in the *Cytbc*₁ (section 3.4) were replaced by alanine residues (6G6A). Moreover, substitutions were created near 2Fe2S (S128 and W163); and seven C-terminal residues were deleted (G171och). The 6G6A and G171och mutations affect highly conserved segments in the chloroplast ISP, but no phenotype was found in these lines. Although extensions of up to five residues or deletion of one residue in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency, deletion of just three residues (Δ3G) dramatically decreased reaction rates by a factor of ~10. Overall, these findings indicate that the chloroplast ISP hinge is also very flexible⁶⁰⁹ and its movement is not limiting for catalysis, consistent with previous conclusions in the case of *Cytbc*₁ (see section 3.4).

Only one mutant has so far been reported to affect PQ(H₂) binding to the Qp site in plants. This mutant (pgrl1)⁶¹⁰ was obtained by random mutagenesis and bears a point mutation in the ISP (P194L). Although the mutation does not affect photosynthetic electron transport at low light intensities, it drastically affects thermal dissipation of absorbed light energy in high light, suggesting that Pro194 may affect H⁺/e⁻ coupling in the *Cytb*₆f. Single amino acid substitutions in *Cytb*₆ at the positions D148, A154, and S159 were generated in the cyanobacterium *Synechococcus* sp. PCC 7002. Mutant phenotyping revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and induction of myxothiazol sensitivity in the D148G mutant, confirming the role of these residues in PQ(H₂) binding and inhibitor sensitivity of the complex⁶¹¹.

## 7.4 Catalytic and side reactions at the QH₂-oxidation site of Cytochromes bc

The oxidant-induced reduction of *b* hemes in sub-mitochondrial particles is an intriguing phenomenon first reported by Chance in 1952 during II International Congress of Biochemistry in Paris (see reference ⁶¹² and citations therein). This phenomenon was difficult to explain until a concept of a semiquinone-mediated electronic bifurcation was introduced by Wikstrom & Berden in 1972.²⁰². The central role of a semiquinone in the mechanism of bifurcation was further developed by Peter Mitchell in the frames of his original idea of so-called proton-motive Q cycle mechanism. ³² This groundbreaking concept of electronic bifurcation, inherent to this mechanism, postulated that QH₂ at the Qo/Qp site undergoes two-electron oxidation reaction but routes for these two electrons are obligatorily different and very efficient. This entails the first electron being transferred to the oxidized
2Fe2S center (the first cofactor of the high-potential chain) while the second electron is transferred exclusively to heme $b_L/b_p$ (the first cofactor of the low-potential chain) see section 3.2.

It is assumed that the mechanism of electron bifurcation is sequential, and involves formation of a transient semiquinone intermediate. While the time between the first electron transfer from QH₂ to 2Fe2S and the second electron transfer from SQ to heme $b_L/b_p$ remains unknown, it seems reasonable to assume that it is not shorter than the time required for electron tunneling from the SQ to heme $b_L/b_p$.

According to Mitchell's principles QH₂ must donate its two electrons to two different chains of cofactors and the two redox couples involved in the first and the second electron transfer – SQ⁻/QH₂ and Q/SQ⁻ couples, respectively, must therefore possess different redox midpoint potentials³², as originally discussed by Wikstrom & Berden in 1972.²⁰² It has been proposed that the $E_m$ of the SQ⁻/QH₂ redox couple is very positive, and comparable to the high potential acceptor (2Fe2S and heme $c_1/f$), while the Q/SQ⁻ is expected to be a more reducing couple and therefore able to donate electrons to the low-potential chain (hemes $b_L/b_p$ and $b_H/b_n$). This separation of $E_m$ values (the split) of the two redox couples implies that the stability constant for the SQ⁻ intermediate at the Q₀/Qₚ site must be very low and hence, detection of such an intermediate by spectroscopic methods might be difficult, especially under equilibrium conditions⁴⁸⁷,⁴⁸⁸. Indeed, until recently, the semiquinone intermediate state associated with the operation of the Q₀/Qₚ site could not be experimentally detected (see section 7.6).

<figure><img src="image_32.png" /></figure>

**Figure 28.** Energy diagram of electron transfer through cofactor chains in Cytbc₁. Horizontal black lines mark $E_m$ values of the respective oxidized/reduced couple and are labeled with an orange letter in bracket. Gray gradient denotes a split in potentials between Q/SQ⁻ and SQ⁻/QH₂ couples at the Q₀ site. The average $E_m$ value of membranous Q/QH₂ pool is
shown in blue. Red and green solid arrows show endothermic and exothermic reactions, respectively. Green dashed arrows show exothermic reaction to the heme states for which $E_m$ are not directly measurable by equilibrium redox titration. Gray horizontal lines show a possible $E_m$ values of hemes $b_L$ and $b_H$ modified by Coulombic interactions between the hemes (assuming the same energy of interactions ~ 80 mV for $b_L$ and $b_H$^{133,206}$), that are not accessible for direct measurement. Vertical double arrows denote the changes in $E_m$ depending on the redox state of the coupled heme. Cytc and Fe-S denote oxidized/reduced couples of Cytc₂ and 2Fe2S; $b_L(b_H^{ox})$ and $b_L(b_H^{red})$ denote oxidized/reduced couple of heme $b_L$, at a time when heme $b_H$ is oxidized and reduced, respectively. $b_H(b_L^{red})$ and $b_H(b_L^{ox})$ redox couple of heme $b_H$ at a time when heme $b_L$ is reduced or oxidized respectively. Note that this scheme does not necessarily apply for Cytb₆f.

### 7.4.1 Forward, reverse and short-circuits reactions at the Q₀/Qₚ site

A simple diagram of $E_m$ values taken from *Rhodobacter capsulatus* Cytbc₁ with associated $\Delta G$ changes due to electron transfer through the cofactor chains is shown in Fig. 28. As originally proposed by Mitchell, there is a large, albeit unknown, split between $E_m$ values (here ~800 mV) of the Q/SQ⁻ and SQ⁻/QH₂ couples, and this split depends on the stability constant of the SQ⁻. This makes the first electron transfer from QH₂ to 2Fe2S essentially endothermic and requires an energy debt for electron transfer to the acceptor of a more negative redox potential (2Fe2S) than the donor (QH₂). This energetic debt is repaid during the second electron transfer from SQ⁻ to heme $b_L/b_p$. The forward reactions, meaning the net electron transfer from QH₂ to Cytc, is continued as long as there is a difference (driving force) between the $E_h$ of membranous Q/QH₂, the *p* side acceptor pools (Cytc, PC or Cytc₆) and all the cofactors of Cyt-bc complexes. Nevertheless, it has been shown by several experiments that under specific conditions Cytbc₁ has the capacity to catalyze a fast, reverse reaction of reduction of Q to QH₂ at the Q₀ site. Such a process was observed in *in vitro* studies of Cytbc₁ reconstituted into liposomes⁶¹³ and also proposed to occur *in vivo* to support the growth of the chemolitotrophic bacteria *Thiobacillus ferrooxidans* using Fe²⁺ ions as a source of electrons⁶¹⁴. This suggests that the reactions at the Q₀ site are rapidly and efficiently reversible, where “efficient” means that these reactions do not lead to significant energy-wasting side reactions (short-circuits or leaks). Indeed, light-induced electron transfer measurements using cofactor knockouts of bacterial Cytbc₁ to dissect out individual electron transfer steps, demonstrated that the reactions of the Q₀ site are reversible on a catalytically-relevant timescale (milliseconds)⁶¹⁵. Fast reversibility of these reactions makes an understanding of the energetic efficiency of electronic bifurcation conceptually challenging. As long as one forgets about the reversibility, the diagram in Fig. 28 simply explains the energetic efficiency of electron transfer onto two different chains of cofactors. However, the fast reversibility of reactions at the Q₀/Qₚ site means that the enzyme can potentially catalyze energy-wasting short circuit reactions²⁵⁹,⁶¹⁵. Four possible short-circuit reactions of the Q₀/Qₚ site are shown in figure 29.
<figure><img src="image_33.png" /><figcaption>**Figure 29.** Scheme showing 4 types of short circuit reactions that are theoretically possible during the catalytic cycle of Cytb*bc*₁ and Cytb*6f*²⁵⁹. Red arrows show energy conserving ET while blue arrows shows energy-wasting short circuiting ET. Numbers denote the order of ET events. Short circuits start with QH₂ (SC1 and SC2) or Q (SC3 and SC4) present at the Q₀/Qₙ site. Gray rhombuses denote hemes and white-yellow circles show 2Fe2S. Scheme does not consider redox states of cofactors. Dashed arrows and 3 positions of 2Fe2S denote movement of the ISP-HD (necessary to complete SC1). See text for details.</figcaption></figure>

The first type of short circuit (SC1) is a reaction in which two electrons from QH₂ are transferred to the high potential chain according to the following reaction sequence:

$$ 
\begin{aligned}
\mathrm{QH_2} + 2\mathrm{Fe2S}^{\mathrm{ox}} &= 2\mathrm{H}^+ + 2\mathrm{Fe2S}^{\mathrm{red}} + \mathrm{SQ}^- \\
2\mathrm{Fe2S}^{\mathrm{red}} + c_1^{\mathrm{ox}}/f &= 2\mathrm{Fe2S}^{\mathrm{ox}} + c_1^{\mathrm{red}}/f \\
\mathrm{SQ}^- + 2\mathrm{Fe2S}^{\mathrm{ox}} &= 2\mathrm{Fe2S}^{\mathrm{red}} + \mathrm{Q}
\end{aligned}
$$

SC1 would be very exothermic since the electron transfer would be associated with a large potential energy change due to the transition from state [b] to [e] in figure 28. Despite the fact that SC1 is thermodynamically very favorable, it is avoided relatively easily, due to the requirement for the movement of ISP-HD (see section 3.4) during transfer of electrons from 2Fe2S to heme c₁ or f. Through this constrained diffusion of ISP-HD, the distance between SQ⁻ and the cluster increases significantly, slowing down any possible second electron transfer from the semiquinone to 2Fe2S. In the absence of ISP-HD in close proximity, the unstable SQ⁻ therefore pushes the second electron onto heme bₗ/bₚ. SC1 decreases the proton transfer efficiency across the membrane: it yields +2H⁺ₚ / 0H⁺ₙ per oxidation of one QH₂, while an energy-conserving Q cycle yields +4H⁺ₚ / -2H⁺ₙ per 1 QH₂.

In contrast to SC1, the second type of short-circuit reaction (SC2) is extremely difficult to suppress without involvement of any additional mechanisms to lower the probability that it occurs. SC2 is a mixture of partial reactions of the forward and the reverse catalysis performed by the Q₀/Qₚ
site and is a result of the following reactions (for simplicity in the following schemes $b_L$ and $b_H$ also represent hemes $b_p$ and $b_n$, respectively):

$$QH_2 + 2Fe2S^{ox} = 2H^+ + 2Fe2S^{red} + SQ^- \\ b_L^{red} + SQ^- + 2H^+ = b_L^{ox} + QH_2$$

SC2 may take place when $QH_2$ undergoes oxidation by $2Fe2S$ as a part of the chemistry of the normal forward reaction, while heme $b_L/b_p$ is reduced and unable to accept electron from $SQ^-$. In this situation, heme $b_L/b_p$ can then donate an electron to $SQ^-$, reforming $QH_2$, as this reaction is also much more energetically favorable than any possible forward electron transfer from heme $b_L/b_p$ to heme $b_H/b_n$ (compare the difference in energy associated with [b] to [a] and [b] to [c] transition in fig. 28).

Energy conversion of this process is compromised as this short circuit gives the same stoichiometry as SC1 ($+2H^+_p / 0H^+_n$ per 1 oxidized $QH_2$), assuming that every SC2 is preceded by $QH_2$ oxidation.

Conditions in which SC2 could take place are created not only when an inhibitor such as antimycin, blocks electron transfer from heme $b_H$ to Q at the $Q_i/Q_n$ site, but also during reverse electron transfer from $QH_2$ bound at the $Q_i/Q_n$ site, which is a natural consequence of the physiological reverse catalysis of Cyt-$bc$. It has been demonstrated (see section 8), that electrons from the $Q_i$ site of one monomer can be transferred through the heme $b_L$ to the other monomer, creating the possibility that heme $b_L$ is already reduced when $SQ^-$ is generated during oxidation of $QH_2$ by $2Fe2S$ at the $Q_o$ site.

The third type of short circuit (SC3) is electron transfer from reduced heme $b_L/b_p$ to Q that is bound concurrently with $QH_2$ to the $Q_o/Q_p$ site, when the oxidized $2Fe2S$ is close to the $Q_o/Q_p$ site. SC3 consists of two sequential reactions: first uphill (transition from states [c] to [b]), followed by downhill ET (transition from [b] to [e] in fig. 28) which can be summarized as follows:

$$b_L^{red} + Q = b_L^{ox} + SQ^- \\ SQ^- + 2Fe2S^{ox} = Q + 2Fe2S^{red}$$

SC3 is quite similar to SC2 and the proton transfer efficiency is the same. The only difference is the fact that SC3 should be favored over SC2 when concentrations of Q in the $Q(H_2)$ pool are higher. However, in the absence of an inhibitor blocking the $Q_i/Q_n$ site or mutation retarding electron transport from heme $b_H/b_n$ to the $Q_i/Q_n$ site, SC3 seems to be less probable than SC2.
The last type of short circuit (SC4) is the two-electron reduction of Q at the Qo/Qp site by the hemes bL/bp and bH/bn of the low-potential chain without involvement of the 2Fe2S. SC4 can be summarized as follows:

$$Q + b_{L}^{\text{red}} = SQ^{-} + b_{L}^{\text{ox}} \\ b_{H}^{\text{red}} + b_{L}^{\text{ox}} = SQ^{-} + b_{H}^{\text{ox}} + b_{L}^{\text{red}} \\ SQ^{-} + b_{L}^{\text{red}} = QH_{2} + b_{L}^{\text{ox}}$$

According to the modified Q cycle³⁹, SC4 is obviously considered as the energy-wasting reaction since it diminishes the number of protons transferred across the membrane. However, calculations of the number of protons that are transferred per oxidized QH₂ under condition in which SC4 takes place is more complex than in the case of SC1, SC2 and SC3.

Let us first consider 4 cycles of QH₂ oxidation steps according to the energy-conserving Q cycle. If the modified Q cycle is operating, this will lead to 8H⁺ released to the p side, 4H⁺ taken from the n side and the net consumption of 2 QH₂ molecules from the Q(H₂) pool. The overall stoichiometry of this modified Q cycle is +8H⁺/p-/4H⁺/n and -2QH₂/+2Q in the Q(H₂) pool.

Now consider a similar sequence of 4 QH₂ oxidation reactions but with a single SC4 reaction occurring in the middle of the process: 2 QH₂ oxidations – SC4 reaction – another 2 QH₂ oxidations. For clarity let us divide the process into three parts. In the first part, 2 QH₂ oxidations yield +4H⁺/p-/2H⁺/n and -1QH₂/+1Q. During the second part SC4 gives -2H⁺/p/+2H⁺/n and +1QH₂. During the third part another 2 QH₂ oxidation take place yielding the same +4H⁺/p-/2H⁺/n and -1QH₂/+1Q as in the first part. Addition of all products from these three parts gives a net stoichiometry of +6H⁺/p-/2H⁺/n and -2QH₂/+2Q in the Q(H₂) pool, which means that 1 occurrence of SC4 per 4 oxidized QH₂ decreases the number of protons released to the p side from 8 to 6. This would give an average +3H⁺/p-/1H⁺/n per 1 QH₂ oxidized during this sequence vs +4H⁺/p-/2H⁺/n per 1 QH₂ in the Q-cycle.

### 7.4.2 The possible physiological meaning of short-circuits

In general, short-circuit reactions decrease the efficiency of energy conversion by Cytbc and the probability of their occurrence should be minimized or slowed down by two or three orders of magnitude in comparison to the productive forward and reverse reactions. It must be emphasized however, that it is still possible for some of the SC reactions to be catalyzed under specific conditions, such as severe mutation or presence of inhibitors which preclude normal catalysis. In fact it has been proposed that the reactions of SC2 could be a potential mechanism to support photosynthetic growth of green alga *Chlamydomonas reinhardtii* strains, in which normal function of Cytb6f is disabled by mutation to replace the heme bH ligand - histidine 202 with glutamine⁶¹⁶.
The reactions of SC4 could also be potentially considered as desirable for electron recirculation back to the PQ(H₂) pool in, for example, photosynthetic CET at the level of Cytb<sub>6f</sub> (see section 4.3). Assuming alternating reactions of oxidation of PQH₂ and reduction of PQ at the Qₚ site, using electrons delivered through the low-potential chain from the Fd/NADPH⁺ pool, the overall proton pumping efficiency would be significantly increased yielding +6H⁺<sub>p</sub>/-4H⁺<sub>n</sub>, -1PQH₂ in the Q(H₂) pool and -2 Fd from the Fd/NADPH⁺ pool. This would contribute to a much higher proton gradient per oxidized PQH₂ than +4H⁺<sub>p</sub>/-2H⁺<sub>n</sub> per 1 PQH₂ resulting from the modified Q cycle mechanism. However, a possible involvement of SC4 in the mechanism of CET around Cytb<sub>6f</sub> and PSI has not been proposed earlier and thus has never been tested experimentally.

Another potential physiological implication of short-circuits relates to the concept of kinetic competition between short-circuits and the leak of electrons (i.e. reactions that may lead to superoxide production). The completion of any type of short-circuit reaction retains electrons within the enzyme, therefore diminishing the probability of a leaks. Thus, this could be considered as a possible means of protection against unwanted release of superoxide, which might be of importance in the context of a postulated signaling role for superoxide release from the Q<sub>o</sub>/Q<sub>p</sub> site (see section 7.5).

### 7.4.3 Short-circuit suppression mechanisms

As indicated in section 7.4.1, understanding the fast reversibility of electron transfer in Cytb<sub>c</sub> and the efficient suppression of short circuit reactions is conceptually challenging<sup>259,615,617,618</sup>. Direct electron transfer between the low- and high-potential chains is significantly limited by the 2.3-nm distance between 2Fe2S and the ring of heme b<sub>L</sub>/b<sub>p</sub><sup>259</sup>. Such a large spatial separation slows down electron transfer to a timescale of seconds, minimizing the probability of its occurrence on a catalytic time scale. However, the problem appears when a redox active molecule such as quinone (QH<sub>2</sub>, Q or SQ<sup>-</sup>) is bound at the Q<sub>o</sub>/Q<sub>p</sub> site and cuts the 2.3-nm distance into two much shorter distances, each being well below the 1.4 nm limit<sup>56</sup>. This means that any type of quinone molecule bound in the site (whether it is the substrate, product or intermediate of the QH<sub>2</sub> oxidation) can in principle act as mediator, to significantly enhance electron transfer between the low- and high potential chains.

To explain the general lack of experimentally detected short circuits several “gating” mechanisms were constructed. The problem is however, that in most cases the gating mechanisms are either very effective at suppressing short-circuits, therefore sacrificing reversibility, or if they allow reversibility they are prone to some short circuits<sup>259,615</sup>. It appears that to explain how the Cytb<sub>c</sub> can accomplish full and rapid reversibility and short circuit suppression while limiting superoxide production, elaborate mechanisms involving more than a single gating factor are needed.
The necessity for elaborate gating is a consequence of the sequential mechanism that is considered to drive the reaction at the Q<sub>o</sub>/Q<sub>p</sub> site, which is obliged to form SQ<sup>-</sup> as a true intermediate of the two-electron reactions of bifurcation. This mechanism assumes formation of SQ<sup>-</sup> at the Q<sub>o</sub>/Q<sub>p</sub> site, albeit at low occupancy<sup>224</sup>. It is generally in line with many observations that the Q<sub>o</sub>/Q<sub>p</sub> site can, under specific conditions, generate superoxide and also with recent experiments reporting detection of a SQ<sup>-</sup> intermediate at this site (see section 7.5 and 7.6 and references there). The sequential mechanism is also in agreement with the chemical properties of quinones and their ability to take part in single-electron redox reactions. However, the major problem with sequential electron transfer is its susceptibility to short-circuiting.

As an attractive alternative and seemingly the simplest solution to the short-circuit problem, a concerted reaction was also proposed for the Q<sub>o</sub>/Q<sub>p</sub> site. This reaction, based on a fundamentally different mechanism, assumes that the reaction takes place through a transition state devoid of semiquinone character. In the following sections we will first reflect on the concerted mechanism before discussing the various possible gating mechanisms related to the sequential mechanism of the bifurcation.

*Short-circuit-proof and leak-proof concerted reaction*

The “concerted” reaction initially proposed for the Q<sub>o</sub>/Q<sub>p</sub> site is ‘thermodynamic’ cooperativity of the bifurcation reaction, in which electron transfer is sequential, but with a transient semiquinone state that never accumulates enough to be observable (i.e. reduction and oxidation of reaction partners may appear concurrent)<sup>155,619,620</sup>. Together with the kinetic resolution of millisecond reversibility of all electron transfer steps within Cytbc<sub>1</sub>, including the reactions at the Q<sub>o</sub>/Q<sub>p</sub> site, the “concerted” reaction was introduced as one of the possible mechanism allowing reversible operation of this site while preventing all short-circuits (the other mechanism introduced in parallel was a “double-gating”, as described below)<sup>259,615</sup>. The term “concerted” was regarded as a “kinetic” definition of two events taking place without time for significant atomic rearrangement. This concept recognized that all short-circuits would be efficiently suppressed if QH<sub>2</sub> oxidation (forward catalysis) and Q reduction (reverse catalysis) took place as a virtually single step involving 2 electron and 2 proton transfers. Suppression would then simply be a consequence of forbidding one-electron transfer reactions, of which all short-circuits are composed (see Fig. 29)<sup>155,259,615,619,620</sup>.

It follows that if the concerted reaction is the catalytic mechanism at the Q<sub>o</sub>/Q<sub>p</sub> site, the SQ<sup>-</sup> intermediate would not be formed at all during either QH<sub>2</sub> oxidation or Q reduction as its lifetime is shorter than the time needed for nuclei reorganization of molecules taking part in the electron transfer reactions<sup>259</sup>. This mechanism elegantly explains the energetic efficiency of the reversible catalysis of
$Cytbc_1$, but it remains hypothetical. While theoretical and experimental descriptions of kinetically concerted two-electron transfer reactions do exist$^{621,622}$, the chemical and physical details of the any concerted reaction at the $Q_o/Q_p$ site remain unknown, as is the mechanism that would explain how SQ⁻ formation is forbidden.

The concerted mechanism becomes difficult to reconcile with recent experiments reporting the detection of transient SQ⁻ intermediates in the $Q_o/Q_p$ site (see section 7.6). As, the concerted reaction is by definition short-circuit-proof and leak-proof, it eliminates entirely the possibility of superoxide generation. It follows, that another mechanism of SQ⁻ formation by $Cytbc$ would therefore have to be envisaged to explain this activity of $Cyt-bc$, such as electron transfer from reduced heme $b_L/b_p$ to $O_2$, for which there is no evidence.

## Gating mechanisms of sequential reaction

### i) Catalytic switch and $QH_2/SQ/Q$ mobility-driven gating at the $Q_o$ site

The “catalytic switch”, proposed by Brandt et al. before the discovery of 2Fe2S motion can be considered the first gating model$^{57,58}$. This model pointed to a key role of the redox state of the 2Fe2S on the bifurcation reaction. It was formulated based on the difference between the binding constants of two types of inhibitors, stigmatellin and MOA-stilbene to $Cytbc_1$, caused by changes in the reduction state of the 2Fe2S cluster and the discovery that ISP-HD can be found at two different positions. The model assumed that $QH_2$ and Q can interact with oxidized 2Fe2S while SQ⁻ interacts with oxidized heme $b_L$ but not with the reduced 2Fe2S cluster. Thus, electron transfer to 2Fe2S or heme $b_L$ strictly depends on the redox state of these two cofactors forcing the forward bifurcation reaction at the $Q_o$ site$^{57}$.

A somewhat similar model was proposed recently to explain the forward electron transfer in bifurcation through the movement of SQ⁻ at the $Q_o$ site, to drive electrons to the correct chain. This model was also based on structural studies of stigmatellin binding to the $Q_o$ site$^{91}$. It predicts that reduction of the 2Fe2S cluster and formation of semiquinone leads to disruption of the H bond between $RhH156$ ($^{Bt}H161$) which favors the movement of ISP-HD to the c-position (see section 3.4 for details) and a rapid movement of the benzoquinone ring toward the $RhE295$ ($^{Bt}E271$)$^{119,575}$. The latter movement facilitates deprotonation of the semiquinone and therefore increases the rate of electron transfer from SQ⁻ to heme $b_L$ by shortening the distance between the reagents$^{23,596}$. After the second oxidation, Q returns to the niche that is close to the 2Fe2S cluster. In this way the redox state of Q and heme $b_L$ determine the direction of electron transfer at the $Q_o$ site. These two mechanisms: termed “catalytic switch” and “SQ motion” do not protect the enzyme from performing SC2 or SC4.
Furthermore, they do not describe the proton/electron sequence of the reverse direction and, as discussed in ²⁵⁹,⁶¹⁵, they are not easily reversible.

### ii) Surface-modulated motion switch of Cytbc₁

Another gating mechanism proposes that the movement of ISP-HD after the 2Fe2S cluster is reduced is coupled to the oxidation of heme $b_L$ by heme $b_H$²¹,⁷⁸,⁸⁹,⁶²³. The authors proposed that electron transfer from heme $b_L$ to heme $b_H$ triggers some conformational changes in the region of Cytb⁸⁹ responsible for docking the ISP-HD, releasing this domain from the $Q_o$ site and then releasing Q and binding $QH_2$⁷⁸. Although, this mechanism allows the reversible reactions at the $Q_o$ site, some short-circuit reactions (SC2, SC3 and SC4) are still possible. Furthermore, this model predicts, that the presence of Q at the $Q_o$ site would favour occupation of positions remote from the $Q_o$ site by the ISP-HD. However, this seems at odds with the experimental observation that when Q is bound at the $Q_o$ site it leads to EPR spectra typical of 2Fe2S interacting with Q for the whole population of the reduced 2Fe2S (see section 3.4 for details).

### iii) Proton gated affinity change and proton gated charge-transfer mechanisms

The “proton gated affinity change” mechanism was formulated on the basis of the electrochemical properties of 2Fe2S, in particular the difference between the pK value of the $RhH_{156}(B^tH_{161})$ for the reduced and oxidized 2Fe2S⁵⁷⁴. According to this mechanism, either $QH_2$ binds to the deprotonated histidine or $QH^-$ binds to the protonated histidine. The first electron transfer to 2Fe2S then drastically increases affinity of $SQ^-$ for the reduced 2Fe2S, leading to a positive shift in the redox potential of the 2Fe2S in the $2Fe2S^{red} – H^+ – SQH$ complex. Such a stabilization should lead to a relatively large occupancy of $SQ^-$ at the $Q_o$ site. However, when this proposal was framed, experiments had thus far failed to detect any $SQ^-$ at the $Q_o$ site. This discrepancy was explained on the ground of strong antiferromagnetic coupling between $SQ^-$ and $2Fe2S^{red}$ leading to disappearance of the EPR signal.

Evoking antiferromagnetic coupling as a reason for the failure to detect an EPR signals implies that the energy of such interactions must be very high to depopulate the paramagnetic, excited state at temperatures > 10 K (typical experimental conditions). However, this kind of strong spin-spin exchange is very unlikely for most bonds through which $SQ^-$ could interact with 2Fe2S, including a single H bond. Interestingly, experiments performed 16 years after the initial formulation of the “proton gated affinity change” mechanism established that a spin-spin exchange interaction between $SQ^-$ and reduced 2Fe2S does occur, but that the energy of such coupling is very low in comparison to
the thermal energy of experimental EPR conditions; and that this state can be detected by EPR (this is described in detail in section 7.6).

As described in section 7.2, several experimental results have been interpreted as being indicative of binding two substrate molecules to the Q₀ site at the same time. This concept was also used to formulate a “proton gated charge-transfer mechanism” in which a QH₂/Q pair must first undergo two deprotonation steps followed by electron transfer to 2Fe2S and heme bL⁵⁸⁶. The two proton-gated mechanisms allow reversibility of the Q₀ site but do not prevent SC2, SC3 nor SC4.

#### iv) Double-gating mechanism

More recent models of bifurcation take into account the fact that a single gating step is generally not sufficient to suppress all types of short-circuits for reversible sequential bifurcation.

The first model of this type was the “double gating” mechanism, introduced as an alternative to the concerted mechanism in providing a means for short-circuit-proof and efficient reversible operation of the Q₀ site. It evoked the concept that changes in protonation/deprotonation of water molecules and/or amino acid side chains near the Q₀ site occur upon changes to the redox state of immediate cofactors, 2Fe2S and heme bL, in order to specifically modulate barriers in the sequential reaction ²⁵⁹,⁶¹⁵. Double gating allows QH₂ oxidation only when both 2Fe2S and heme bL are oxidized and allows Q reduction only when both 2Fe2S and heme bL are reduced. This would be accomplished by raising the barrier for SQ⁻ formation when QH₂ is bound at the Q₀ site (involving a SQ⁻/QH₂ couple) when heme bL is reduced and the 2Fe2S cluster is oxidized, or by lowering that barrier when both cofactors are oxidized. This would prevent SC1 and SC2 (Fig. 29). On the other hand, the barrier for SQ formation when Q is bound at the Q₀ site (involving Q/SQ⁻ couple), would be raised when heme bL is reduced and the 2Fe2S cluster is oxidized, or the same barrier is lowered when both cofactors are reduced. This would prevent SC3 and SC4.

#### v) Logic-gating mechanism

A “logic-gating” mechanism is quite similar to the “double gating” mechanism. It also evokes a concept of changes in protonation/deprotonation of water molecules and/or amino acid side chains near the Q₀ site upon changes in the redox state of the immediate cofactors 2Fe2S and heme bL. The model proposes that crucial elements for energy-conserving redox reactions depend on specific H-bond formation in the Q₀ site ⁵⁹⁷. Binding of QH₂ requires $^{Rh}H156(^{Bt}H161)$ and $^{Rh}E295(^{Bt}E271)$ to be in deprotonated states, which is possible when 2Fe2S and heme bL are oxidized. Otherwise, reduction of 2Fe2S and heme bL induces protonation of the N<sub>t</sub> atom of the histidine and the carboxyl group of the glutamate, allowing binding of Q to the Q₀ site. Such a mechanism imposes constraints on the
direction of reaction, as QH₂ binds predominantly when both 2Fe2S and heme b_L are oxidized and Q binds predominantly when both 2Fe2S and b_L are reduced. This mechanism, like the double-gating mechanism, protects against all short-circuits while still allowing reversibility²⁵⁹. It is also generally consistent with the fact that reduction of 2Fe2S is associated with a shift in the pK value of the 2Fe-2S protein from about 7.6 to more than 10⁵⁹⁴ and is in line with the observation that Q bound at the Q₀ site strongly influences EPR spectra of the 2Fe2S (see section 7.2 for details). In this model a tentative coupling between hemes b_L and the Glu residue exists and thus the reduction of heme b_L would be associated with protonation of the Glu carboxylic group while oxidation of the heme would induce deprotonation of the same side chain. However, experimental results on a series of mutants that replace $^{Rh}$E295($^{Bt}$E271) with other amino acids revealed only minor effects on the redox midpoint potential of heme b_L⁵⁷⁹,⁵⁹⁸. Also, equilibrium redox titrations of the bacterial chromatophore Q(H₂) pool, made by monitoring changes in the gₓ EPR transition of the 2Fe2S, showed that the reduced cluster is still able to interact with QH₂ under conditions in which heme b_L remains oxidized. The experimental detection of Q bound to the Q₀ site under conditions of reduced heme b_L is not feasible due to large difference in Eₘ values of the Q/QH₂ couple and heme b_L.

In this mechanism water dipoles are proposed to be the crucial elements that stabilize Q or QH₂ binding when 2Fe2S and heme b_L are either both reduced or both oxidized, respectively.

### vi) *Coulombic gating*.

When the rapid reversibility of reactions at the Q₀ site was established¹³⁶, it became clear that the “SQ⁻ movement” mechanism (see above) does not explain gating, nor does it prevent all types of short-circuit. In an attempt to modify this mechanism to accommodate reversibility and the prevention of all short-circuits “Coulombic gating” was proposed⁵⁹⁶. The crucial element of this model is an assumption that SQ⁻ is attracted by Coulombic interactions, and thus moves toward the oxidized heme b_L, so increasing the electron transfer rate. On the other hand, when SQ⁻ is formed while heme b_L is reduced, then it is repelled, moving toward the 2Fe2S and decreasing the probability of electron transfer from heme b_L to SQ⁻. Although, this mechanism seems to explain the limited efficiency of short-circuit SC2, it is still not clear how it would allow rapid reversibility of the Q₀ site.

*Current status and emerging concepts of short-circuit suppression.* Despite many proposals and the various models outlined above, the exact mechanism of gating and reversibility at the Q₀ site that minimizes energy-wasting reactions is still not known. It appears that each of the mechanisms presented is able to explain the bifurcation reaction under certain specific conditions, but at the same time they may not be sufficient to explain observations under other conditions, or to provide a detailed
molecular basis, or be fully consistent with the ensemble of existing experimental data. Our intention
in this review is not to favor any particular mechanism. Rather, in view of their diversity and general
complexity, and also given that no obvious elements unifying them into one common mechanism can
be proposed, we argue that the bifurcation at Q₀ site is far from being satisfactorily understood and
still deserves extensive experimental and theoretical exploration. This is particularly the case for the
Cytb₆f, where the situation is further complicated by the possible injection of an additional electrons
from the *n* side through the c-heme to heme *b*ₙ.

It is worth hypothesizing that the “true” mechanism of energy-conserving reversible
bifurcation could be a process to which several of the many different gating mechanisms proposed
by different authors could contribute, depending on the starting conditions. We also anticipate that
the transition from forward to reverse electron transfer at the Q₀ site may not be a simple reversal of
the proton/electron transfer, and that dynamic events that start the reverse reaction may differ from
the final step of forward reaction. It could very well be that the stability constant of the SQ⁻ formed
during oxidation of QH₂ is different from the stability constant of SQ⁻ formed by reduction of Q. We
believe that the key players are likely to be protons, as different proton configurations could favor
different stabilities of the SQ⁻ formed during forward and reverse reactions. However, the proton
events and paths for uptake and release have not been resolved yet.

7.5 Superoxide generation at the Q₀/Qₚ site

The operation of any electron transfer chain (ETC) in the presence of oxygen is connected to
the generation of superoxide radical. *In vivo*, superoxide is quickly converted into H₂O₂ in a reaction
catalyzed by superoxide dismutase. This is presumably why first mentions of ETC-derived radicals
were related to H₂O₂ and not superoxide. Indeed, the first information about reactive oxygen species
(ROS) in mitochondria showed mitochondrial levels of H₂O₂ at 1-2% of total O₂ uptake in state 4 of
respiration (the state not coupled to ATP synthesis)while the rate of H₂O₂ generation in state 3 of
respiration (the state associated with ADP to ATP conversion) was negligible⁶²⁴,⁶²⁵. It was also noticed
that antimycin enhances production of H₂O₂ in intact mitochondria ⁶²⁶,⁶²⁷.

*Semiquinone at the Q₀/Qₚ site as electron donor to molecular oxygen* The first reports on
superoxide production in antimycin-treated submitochondrial particles to suggest that ROS are
Cytbc₁-derived appeared in Boveris and Cadenas work in 1975 ⁶²⁸⁻⁶³⁰. Over the next few years, it was
established that the origin of ROS in antimycin-inhibited Cytbc₁ of submitochondrial particles is the
redox reaction in the UQH₂ oxidation site ²⁵⁴,⁶³¹. Using Cytbc₁ isolated from yeast, it was also shown
that inhibition with antimycin A as well as with myxothiazol which binds to the proximal niche of the
Q₀ site, leads to production of ROS at the Q₀ site⁶³²,⁶³³. The link between superoxide production and the action of Q₀/Qₚ site is consistent with the widely-accepted notion that a highly unstable SQ⁻ at the Q₀ site (SQ₀) is a direct one-electron donor to oxygen²²⁴.

In view of the reversibility of the catalytic reactions in the Q₀ site, there are two possible reactions that can lead to the formation of semiquinone at this site: one electron oxidation of QH₂ by the oxidized 2Fe2S cluster (the “semiforward” reaction)⁶³²,⁶³⁴ or one electron reduction of quinone by heme bₐ (the “semireverse” reaction,⁶³⁵,⁶³⁶. It therefore follows that there are two possible reaction sequences leading to ROS production by the Q₀ site. These sequences involve either semiforward or semireverse reaction followed by electron transfer from the formed SQ⁻ to molecular oxygen. Both sequences require the initial presence of reduced heme bₐ which either blocks the heme bₐ from accepting an electron from SQ⁻ formed via the semiforward reaction or donates an electron back to Qₚ bound at the Q₀ site, forming SQ⁻ via the semireverse reaction. This requirement is generally consistent with the observation that ROS are detected when electron transfer between heme bₐ and heme bₚ is impeded by blocking the Qᵢ site with antimycin A or by an increase in membrane potential⁵⁷²,⁶³⁷.

In the currently discussed mechanisms of ROS production by the Q₀/Qₚ site, both reaction sequences (i.e. involving semiforward or semireverse reaction for semiquinone formation) are taken into account. Some models propose that only one from these two sequences is responsible for ROS production, while other models consider a contribution from both. Indeed, it seems plausible that the detected ROS may be a combined product of both mechanisms, but that their relative contributions change depending on the experimental conditions and/or introduced electron transfer barriers within cofactor chains.

The specific rate of superoxide production in Cytb₅f, normalized to the rate of electron transport, is more than an order of magnitude greater than that measured in isolated yeast respiratory Cytbc₁ complex.³⁸ The higher rate of superoxide production in Cytb₅f could be a consequence of an increased residence time of plastosemiquinone/plastoquinol in its binding niche near ISP-HD, resulting from (i) occlusion of the quinone portal by the phytyl chain of the unique bound chlorophyll, (ii) an altered environment of the proton-accepting glutamate believed to be a proton acceptor from semiquinone, or (iii) a more negative redox potential of the heme bₚ on the electrochemically positive side of the complex.³⁸

*"The semireverse-Rieske off" model of superoxide production*
The mechanism involving the semireverse reaction (heme $b_L$ to Q electron transfer) is a relatively new phenomenon that emerged from the observation that ROS production exhibits a bell-like shape dependence on the redox state of the Q(H₂) pool. This means that maximal production of ROS occurred when the pool was partially oxidized, which was taken as an indication that the semireverse reaction, for which the substrate is Q (not QH₂), is involved in superoxide generation¹²⁰,⁶³⁶.

A contribution of the semireverse reaction in ROS generation was also established in studies using mutated forms of the bacterial *Cytbc*₁, which introduced limitations in electron transfer at the level of different cofactors within the low and high potential chains ¹⁰⁷,¹²⁰,⁶³⁵. Furthermore, these studies revealed an important constraint associated with the kinetic effects of the motion of ISP-HD. In the proposed model, production of radicals depends on the position of the ISP-HD with respect to the Q₀ site. Mutants with the insertion of a single or two alanine residues (+1Ala, or +2Ala, respectively) at the neck region of ISP, which increases the probability of ISP-HD being located in close proximity to the Q₀ site ⁹⁰,¹⁰⁷, were found to be less prone to ROS production in antimycin A inhibited *Cytbc*₁ ¹⁰⁷,⁶³⁵ (Fig. 30). To explain this observation, it was proposed that molecular oxygen competes kinetically for electrons with cofactors of the Q₀/Qₚ site. When ISP-HD is present at the Q₀/Qₚ site during SQ⁻ formation, the probability of electron transfer between the 2Fe2S and SQ⁻ is high. The reduced 2Fe2S can then reduce the SQ⁻, reforming QH₂ and completing the reverse reaction, while the oxidized cluster can accept an electron from SQ⁻ and completing short-circuit reaction (SC3 in Fig. 29). However, when ISP-HD occupies positions remote from the Q₀/Qₚ site at the time of SQ⁻ formation, the rate of the electron transfer between SQ⁻ and the 2Fe2S decreases. Under these circumstances, the probability that molecular oxygen outcompetes 2Fe2S for electrons from the SQ⁻ increases, which leads to the release of detectable levels of superoxide from the Q₀/Qₚ site.

Initial studies on the +2Ala mutant were not able to discriminate between the possible contributions of semiforward or semireverse reactions in ROS generation ⁶³⁵. In addition to the suppression of ROS generation in the 2Ala mutant, this study described another mutant, $RhM183L$, that changes one of the axial ligands of heme $c_1$, shifting the $E_m$ of this heme by more than -300 mV. This introduced a large barrier to electron transfer between 2Fe2S cluster and heme $c_1$ without affecting the motion of ISP-HD, causing an increase in ROS production. Given that ROS production is also enhanced when electron transfer barrier is introduced at the level of the low potential chain (addition of antimycin A, a mutation disturbing the oxidation of QH₂ at the Q₀ site, $ScE272Q$ ($RhE295Q$) ⁵⁹⁸ or mutations to change the axial ligands of hemes *b*, $RhH198N$ and $RhH111N$ ⁶³⁸), it appears that in general any significant asymmetry in the overall rate of electron flow between the high- and low-potential chains might make the enzyme more prone to generate superoxide. One
exception to this is if it is caused by arresting the ISP-HD at the Q<sub>o</sub>/Q<sub>p</sub> site. Again, these studies could
not discriminate between the relative contribution of semiforward or semireverse reactions to ROS
generation by the Q<sub>o</sub>/Q<sub>p</sub> site.

Such discrimination became possible in subsequent studies with the use of the 1Ala mutant
and in combination with <sup>Rh</sup>M183K (equivalent of <sup>Rh</sup>M183L described above). These studies clearly
pointed toward the semireverse reaction as the dominant contributor in this process <sup>107</sup>. Mutational
studies analyzing the molecular effects of mitochondrial disease-related mutations further
substantiated the inclusion of both postulates (i.e the one related to involvement of the semireverse
reaction in SQ<sup>-</sup> formation and the other related to the probability of ISP-HD position-dependent
interaction of SQ<sup>-</sup> with oxygen) in what can be referred to as the *semireverse-Rieske off* model of
ROS generation by the Q<sub>o</sub>/Q<sub>p</sub> site.

The <sup>Rh</sup>G167P mutation in *Rhodobacter capsulatus* is an equivalent of human mitochondrial S151P mutation in *Cytb* which was found in patients with exercise intolerance <sup>639</sup>. This mutant exhibits enhanced production of ROS even without antimycin A <sup>120</sup>. In contrast to +nAla insertions, <sup>Rh</sup>G167P influences the motion of ISP-HD in such a way that it tends to occupy positions more remote from the Q<sub>o</sub> site in comparison to WT (see section 3.4) This effect, in agreement with the *semireverse-Rieske off* model, increases the probability of SQ reaction with oxygen (Fig. 30). Furthermore, introduction of the +1Ala mutation to the <sup>Rh</sup>G167P mutant partially compensated the effect of <sup>Rh</sup>G167P, which resulted in diminished ROS production, again consistent with this model. The <sup>Rh</sup>G332D mutation, an equivalent of another mitochondrial disease-related mutation G290D (associated with exercise intolerance in humans <sup>640</sup>), was also found to influence motion of ISP-HD. However, this influence is less pronounced compared to <sup>Rh</sup>G167P, and accordingly the effects on ROS production are more subtle <sup>121</sup>, (Fig. 30).

As introduced in section 7.4.1 the kinetic competition between the leaks of electrons and short-circuit reactions, inherent also to the *semireverse-Rieske off* model, might be of physiological importance. In general, both reactions lead to a drop in the efficiency of electron bifurcation at the Q<sub>o</sub> site, but while short-circuits retain electrons within ETC, leaks generate ROS.

Assuming that high production of ROS is deleterious, in some cases short-circuit reactions
may play a beneficial role in lowering of ROS generation. For various enzymes involved in
bioenergetics it has been postulated that such energetic loss, which minimizes ROS-related damage,
gives the organism a better chance of survival <sup>641</sup>. On the other hand, ROS are also postulated to play
a redox signaling role <sup>642</sup>, thus careful balancing of short-circuits and leaks must be considered as one
of the possible elements of the redox signaling/control systems.
When disabled, the Qᵢ/Qₙ site does not completely inhibit the function of the Q₀/Qₚ site, and the enzyme can still sustain electron flux through the ETC. This residual flux is presumably possible via short-circuit reactions and electron leaks. In Cytbc₁ that flux is low enough to make the enzyme non-functional. Interestingly, in Cytb₆f, a residual flux can sustain the entire photosynthetic chain with the short-circuits that were proposed to act as an “emergency exit” pathway bypassing the Q-cycle and making it dispensable⁶¹⁶.

*Other postulated reactions involving molecular oxygen.* In addition to the reaction of molecular oxygen with SQ⁻ formed at the Q₀/Qₚ site discussed above, it was also proposed that oxygen might act as a redox mediator between QH₂ and heme bₗ⁶⁴³. According to this proposal, heme bₗ would accept an electron from QH₂ via protonated superoxide (HOO) at the Q₀ site, which would facilitate electron transfer within the low potential chain. This, so far purely hypothetical scenario was introduced to explain the observed enhanced enzymatic activity of Cytbc₁ in the presence of oxygen, compared to its activity under anaerobic conditions,

MD simulations showed that O₂ molecules spontaneously diffuse into the Q₀ site of Cytbc₁ and can react with SQ⁻ while forming superoxide ⁶⁴⁴. Molecular oxygen may also occasionally get within 5 Å of the central iron atom of the hemes b>L and b>H. Its presence near the heme b>L raises the question of whether this heme is also an immediate electron donor in the production of superoxide. Indeed, this hypothesis has never been definitively ruled out, and although much less popular compared to models based on SQ⁻, can also be found in the literature ⁶⁴³. It is based on the rationale that the redox potential of heme b>L is sufficiently low to make electron transfer to oxygen theoretically possible ⁶⁴⁵.

In this context an interesting concept concerning the evolutionary adaptation of Cyt-bc complexes to the presence of oxygen was proposed recently by Bergdoll et al ¹⁶⁰. By comparing redox properties of organisms that use different quinones and live in aerobic and anaerobic conditions, they concluded that the Great Oxidation Event occurred about 2.5 billion years ago when the rise of atmospheric oxygen resulted in an up-shift of the ambient redox potential of hemes b and the 2Fe2S cluster. The global response of organisms, which included up-shift of the redox potential of the Q(H₂) pool by 150 mV (menaquinone versus ubi/plastoquinone) and a commensurable increase in the entire set of redox potentials of the cofactors of Cytbc complexes, was aimed to avoid deleterious ROS generation.

A separate question that can be found in the literature is whether the Qᵢ/Qₙ site is also able to produce ROS ⁶⁴⁶. Under high concentrations of QH₂ and in the presence of an inhibitor of the Q₀ site, it is possible to generate a stable SQ⁻ intermediate originating from the reverse reaction at the Qᵢ site
of Cytbc₁ (see section 8.3) ¹⁵¹,⁶⁴⁷,⁶⁴⁸. If Qᵢ/Qₙ-produced ROS have physiological significance, it should be exposed under such conditions. However, measurements conducted in the presence of stigmatellin – a Q₀/Qₚ site inhibitor, did not show statistically significant production of radicals in such a system ⁶³³. As discussed in section 8 Qᵢ-derived SQ⁻ can be stabilized *via* hydrogen bonding to prevent potential reaction of this SQ⁻ with molecular oxygen, as was shown for solution-generated SQ⁻, which was found to be stabilized through hydrogen bonds with the acceptor solvent²⁵³.

*Physiological considerations.* The currently available molecular mechanisms of superoxide generation by Cytbc₁ are derived mostly from analysis of conditions where electron flow through the cofactor chains was severely impeded by various experimentally imposed barriers (inhibitors, mutations). This raises the question whether the natively operating enzyme contributes to ROS generation in living cells, and if so how it does this. Despite some reports that uninhibited Cytbc₁ may produce trace amounts of radicals ³⁸,⁵⁷²,⁶⁴⁹, there are no conclusive data to answer this question in a comprehensive manner. In our view further progress requires development of new spectroscopic approaches that not only allow experiments to be conducted in conditions closer to those encountered by the enzyme in the cells, but also provide high molecular and kinetic resolution of specific electron transfer steps. Nevertheless, several important physiological aspects of cytochrome Cytbc₁-related ROS production by ETC are worth discussing.

It is known that production of ROS is dependent on the respiration rate of mitochondria ³⁷,⁶⁵⁰. Factors enhancing ROS production in mitochondria include high pmf, a high NADH:NAD⁺ ratio, high QH₂/Q ratio, low local O₂ concentration and nearly inactive ATPase³⁷,⁶³⁷,⁶⁵⁰⁻⁶⁵². As a rather general rule, the more reduced the cofactors of ETC are, the more prone ETC is to radical formation. Factors lowering production of ROS include high activity of uncoupling proteins which decrease pmf ⁶⁵³. High respiration rate also lowers the probability of ROS production ³⁷,⁶⁵⁰.

ROS generation is usually expressed in units of s⁻¹ (as the amount of superoxide molecules produced by ETC complex in one second) or as a percentage of ROS production (as % of ETC-derived electrons that pass to oxygen forming superoxide). In state 3 of respiration (upon the addition of ADP) when the rate of oxygen consumption is maximal, the fractional ROS production is negligible. However, in state 4 of respiration (when the ADP levels are depleted) and where respiration chain is slowed down, production of ROS is at the level of 1-2 % ⁶²⁴. Interestingly, expressing production in s⁻¹ shows that the rate of ROS production in state 3 might be even higher than in state 4 ⁶⁵⁴.

The possible influence of physiological factors on ROS generation by Cytbc₁ is perhaps best exemplified by the effects of the membrane potential. In its absence, heme bH is the preferred electron
acceptor within the pair of hemes $b_L - b_H$⁶⁵⁵. In its presence, the apparent midpoint potentials of these two hemes become similar, leading to a more equal distribution of electrons between them ⁵⁷³. Consequently, the reduction level of heme $b_L$ increases which, as observed experimentally, is expected to lead to ROS production by the $Q_o$ site ⁵⁷³.

However, a description of dynamic changes in electron distribution between the *b* hemes appears to be a more complex issue when possible communication between monomers in the dimer occurring at the level of the heme $b_L-b_L$ bridge, is considered (see section 9). Any significant electron transfer between monomers is expected to decrease the reduction level of the *b* hemes (including heme $b_L$) which might decrease the levels of ROS released from the $Q_o/Q_p$ site, in particular if the *semireverse-Rieske off* mechanism is considered.

The physiological role of superoxide released by the components of ETC remains an open issue, especially in view of the suggestions that it may be one of the components of cellular redox signaling ³⁶. Among four ETC complexes (Complex I – Complex IV) it is commonly acknowledged that only two of them generate the majority of ROS produced during the operation of the ETC: Complex I and Complex III (reviewed in ⁶⁵⁰,⁶⁵⁶). These two complexes release superoxide into different compartments of mitochondrion: Complex I frees superoxide into the matrix, while Complex III- releases it into the intermembrane space (IMS). ROS in the IMS are more easily accessible to the cytosol compared to ROS in the matrix, as the former need to cross only the outer mitochondrial membrane while the latter have to cross both inner and outer mitochondrial membranes ⁶⁵⁷.

It should be emphasized that consideration of the role of ROS in cellular signaling should always account for the fact that the level of ROS released into the cytosol from mitochondria is the net result of ROS production and its scavenging inside the organelles.

Superoxide generated in the matrix is efficiently converted into $H_2O_2$ by the highly abundant MnSOD ⁶²⁷ and $H_2O_2$ is further consumed by catalase (CAT) and glutathione peroxidase (Gpx). Estimates indicate that $H_2O_2$ is kept at a low level in the matrix, around $5 \times 10^{-9}$ M ⁶⁵⁸. Supposedly, matrix-generated superoxide and $H_2O_2$ from its dismutation do not leave the matrix ⁶⁵⁷ and thus any putative signal derived from complex I is probably restricted to this compartment.

ROS signaling communication between the mitochondria and cytosol becomes more plausible for superoxide generated by complex III and released to IMS ⁶⁴²,⁶⁵⁹.

In the IMS superoxide can be transformed by CuZnSOD to $H_2O_2$ or, theoretically, scavenged by Cytc. The latter reaction, in view of the ratio between superoxide and the high physiological concentration of Cytc in the IMS (estimated to be in the range of 1 mM ⁶⁶⁰), raises concern about whether the lifetime of superoxide released to IMS would be sufficient to carry out any meaningful
signaling purposes. However, the probability of superoxide oxidation by Cytc may actually not be that high considering the rather low ratio of Cytc to Cytbc₁, (around 2:1 ²⁰⁴, as estimated based on the optical spectrum of reduced mitochondria ²⁵⁴,⁶⁶¹) and the fact that Cytc pool is almost fully reduced in state 3 or 4 of respiration ⁶⁶². While superoxide cannot pass efficiently through the membrane, it can escape mitochondria via voltage-dependent anion channels ⁶⁶³. After spontaneous or CuZnSOD-driven dismutation, superoxide is converted into H₂O₂, and in this form it can reach cytosol via diffusion much more readily. When overactivity of CuZnSOD in the IMS is observed, this leads to overproduction of H₂O₂, which in turn may lead to the formation of oxoferryl Cytc. This may result in the oxidation of cardiolipin and turn on early pro-apoptotic processes ⁶⁶⁴.

Interestingly, until now no one has succeeded in detecting superoxide evolution from Cytbc₁ by spin-trapping. This seems to stem from the methodological difficulty related to the large difference between rate constants for the superoxide reaction with Cytc (k = 2.5x10⁵ M⁻¹s⁻¹, ⁶⁶⁵) and the EPR spin trap DMPO (k = 10 M⁻¹s⁻¹, ⁶⁶⁶). DMPO falls behind oxidized Cytc when competing for superoxide. Cytc is a substrate for Cytbc₁ and achieving measurable level of radicals requires measurements to be done in the presence of high micromolar concentrations of Cytc. In order for DMPO to be able to trap superoxide before it reacts with Cytc, it would need to be in a high molar concentration range, which is unattainable due to its limited solubility.

A

<figure><img src="image_34.png" /></figure>

B

<figure><img src="image_35.png" /></figure>
Figure 30. The „semireverse-Rieske off” model of ROS generation in the Q<sub>o</sub> site. (A) A simplified static scheme showing one of the possible sequences of reactions leading to ROS formation at the Q<sub>o</sub> site: oxidation of QH<sub>2</sub> (“forward” box), electron transfer from heme b<sub>L</sub> to Q (“semireverse” box), electron transfer from SQ to molecular oxygen (“superoxide production” box). In “forward”, and “semireverse” box, white and red rhombus correspond to the redox state of heme b<sub>L</sub> before the reaction (oxidized and reduced, respectively). The reduced heme b<sub>L</sub> necessary to start the semireverse reaction can also be a product of electron transfer from heme b<sub>H</sub> (reaction 2 in SC4 in Fig. 29). Note that the semireverse reaction corresponds to reaction 1 in SC3 and SC4 or reaction 2 in SC2 in Fig. 29 (B) Scheme illustrating that a shift in the equilibrium distribution of the ISP-HD positions from the b-position toward the c-position (from left to right) induced by specific mutations correlates with an increase in the ability of the Q<sub>o</sub> site to generate ROS.

7.6 Semiquinone intermediate at the Q<sub>o</sub> / Q<sub>p</sub> site

When discussing the mechanism of QH₂ oxidation at the Q₀/Qₚ site, SQ is naturally considered
as the transient state of the bifurcation reaction. Over the years the EPR signal related to this state
was expected to be typical of a free radical single-line EPR spectrum at a g position close to 2. The
very first report of detection of a g = 2 signal assigned to semiquinone at the Q₀ site (SQ₀) in
submitochondrial particles from 1981⁶⁶⁷ was invalidated by studies from 1998⁶⁶⁸. Since that time a
notion about SQ₀ being highly unstable and therefore undetectable by EPR has been popular in the
literature until 2007, when two independent groups reported on detection of SQ₀²⁶¹,²⁶² In 2013, three
additional reports presenting g = 2 EPR signals assigned to SQ₀ were published ²⁶³,²⁶⁴,⁵⁹⁰ All these
relatively new reports reopened a discussion on the presence of SQ₀, its properties and its implications
for the mechanism of bifurcation. In general, one would expect the signal to appear under conditions
favoring electron transfer reactions at the Q₀/Qₚ site and to be sensitive to all specific inhibitors of
this site. It should also have paramagnetic properties related to its interactions with other metal
cofactors of the site, the heme bₗ/bₚ and/or 2Fe2S (such as enhanced paramagnetic relaxation rates
compared to a chemically generated SQ in solution). This, however, has not always appeared to be
the case. For this reason, in our view, the issue remains debatable and requires further experimental
exploration. A detailed discussion of different spectroscopic properties and controversies surrounding
the g = 2 signals assigned as SQ₀ is beyond the scope of this review. The reader is referred to a more
detailed review by Pietras et al. ²⁵⁸.

In one of the reports on SQ₀ from 2013, apart from the single-line signal at g ~2, measured at
200 K, a new, unexpected transition with the most prominent line at g = 1.94 in the region of the
spectrum of 2Fe2S was recorded at 20 K ²⁶³ (see Fig. 31). The new signal was highly sensitive to the
inhibitors of the Q₀ site and was transient (i.e. was detected only under non-equilibrium conditions
before the enzymatic reaction of Cytbc₁ with its substrates reached equilibrium). Furthermore it was
observed that g=1.94 at the X band (~ 9.5 GHz) shifts to g=1.96 at the Q band (~ 33 GHz). These
findings, complemented by the simulation analysis of the EPR spectra led the authors to conclude that the g = 1.94 transition belongs to the spectrum originating from SQ₀ coupled by spin-spin exchange interaction with the reduced 2Fe2S (SQ-2Fe2S). An isotropic exchange constant of the interaction was estimated at 3.5 GHz. This was all consistent with the theory of exchange interactions, which assume that in the case of spin-spin coupling with an exchange constant of several GHz, the position and shape of EPR transitions of a system of coupled paramagnetic centers will depend on the microwave band used to make the measurements<sup>669,670</sup>. It is of note that the spin-spin exchange interaction between SQ and the reduced 2Fe2S has already been put forward as an explanation for the failure to detect the g = 2 radical signal of SQ<sup>574</sup>. However, unlike the original proposal, the more recent data do not result from anti-ferromagnetic coupling of high energy.

Further research showed that a g = 1.94 signal of similar amplitude was observed when the samples were prepared under both aerobic and anaerobic conditions<sup>482,671</sup>. This dismissed concerns raised by others<sup>672</sup> that it might have resulted from oxidative damage to the 2Fe2S. This also pointed towards an interesting possibility that the SQ-2Fe2S state is not reactive toward molecular oxygen (see discussion below).

Upon optimization of reagent concentrations the SQ-2Fe2S signal was detected within the timescale of tens to hundreds of milliseconds, which was comparable to the time of a single turnover of enzyme under the applied conditions (inhibition of the Qᵢ site with antimycin).<sup>671</sup> On the other hand, the time scale of SQ-2Fe2S evolution was slower than the time scale of uninhibited enzyme turnover. Remarkably, so far the SQ-2Fe2S state has been found to coexist exclusively with oxidized heme b<sub>L</sub>. All together, these results implicated that the semireverse reaction (heme b<sub>L</sub>/b<sub>p</sub> to Q electron transfer) at the Q<sub>o</sub>/Q<sub>p</sub> site is responsible for initiating formation of the SQ-2Fe2S state. This is partly because the semireverse reaction creates SQ<sub>o</sub> which can undergo spin-spin exchange with reduced 2Fe2S provided that the ISP-HD is docked at the site<sup>263</sup>.

Further studies revealed that the probability of formation of the SQ-2Fe2S state varies in mutants with decreased motion of ISP-HD<sup>671</sup>. It is highest in mutants which have ISP-HD shifted towards the Q<sub>o</sub> site (+1Ala and +2Ala). In contrast, the SQ-2Fe2S state was not observed in the <sup>Rh</sup>G167P mutant, which has the oppositely effect to +1Ala or +2Ala and shows impaired docking of ISP-HD into the Q<sub>o</sub> site <sup>120</sup> (<sup>Rh</sup>G167P mutation).

The various combinations of these mutations have shown that direct interaction between the 2Fe2S and SQ₀ is crucial for formation of SQ-2Fe2S state, and even minimal separation of the cluster from SQ₀ induced by mutation breaks the spin-spin interaction.

The same family of mutants also revealed a clear negative correlation between superoxide production and the ability to form the SQ-2Fe2S state. Based on this observation and the non-
reactivity of SQ-2Fe2S center with molecular oxygen discussed earlier, it has been suggested that the SQ-2Fe2S state may be involved in protection against massive release of superoxide under conditions when electron flow through the Q₀ site is impeded ⁶⁷¹. It should be noted that while the lack of the SQ-2Fe2S state is associated with an increase in the production of superoxide by Cytc₁, a high superoxide production may not necessarily imply lack of the SQ-2Fe2S state. This seemingly paradoxical statement is easily explained assuming that the unstable SQ₀ is a common substrate for production of superoxide and generation of the SQ-2Fe2S state. An increase in the probability of formation of SQ₀ will result in both an increase in superoxide production as well as in more efficient generation of SQ-2Fe2S. Since the SQ-2Fe2S state is nonreactive with oxygen its formation can be regarded as an alternative route to leaks of electrons on oxygen thus it reduces the amount of superoxide production.

The SQ-2Fe2S signal was also detected in enzyme operating with native substrates in membranes of photosynthetic bacteria ⁶⁰⁷. Remarkably, the signal was present only when light-activation of the membranes shifted the redox state of the substrate pools, ubiquione and Cytc₂ pools, to a more reduced and oxidized state, respectively.

Under these conditions, SQ-2Fe2S titrated at higher ambient redox potentials compared to the Q(H₂) pool (see section 4.1), which suggests that the SQ-2Fe2S state is somewhat stabilized at the Q₀ site. The redox potential at which the SQ-2Fe2S amplitude reaches its maximum increased with decreasing pH indicating that a proton is involved in stabilizing the SQ-2Fe2S state. The interaction of SQ₀ through hydrogen bonding with the 2Fe2S coordinating residue $R^{h}H156$, and the role of this interaction in SQ₀ stabilization have been postulated many times ²⁵⁸,⁵⁷⁴,⁵⁹⁶.

Interestingly the SQ-2Fe2S state is not restricted to Cytc₁, but has as it was also observed in isolated spinach Cytb₆f exposed to its substrates, PQH₂ and PC ⁴⁸². In this case however, unlike in Cytc₁, inhibition of the Qn site was not required. This revealed that the probability of SQ-2Fe2S state formation when the enzyme operates without inhibition is higher in the Qp site of Cytb₆f compared to the Qo site Cytc₁. The average redox potential of hemes bₙ and bₚ is probably more negative in Cytb₆f than respective b hemes in Cytc₁, while the redox potential of PQ/PQH₂ couple is higher compared to the potential of the respective UQ/UQH₂ couple. These differences in the redox potentials of b hemes and quinones would foster the semireverse reactions at the Qp site, and thus increase the probability of the SQ-2Fe2S state formation. In addition, electron transfer through heme cₙ in Cytb₆f (not present in Cytc₁) might slow electron flow through the low-potential chain, and this could lead to a more favorable semireverse reaction.

The importance of quinone redox potential to SQ-2Fe2S formation is well supported by the observation that the high-potential Q derivative (DBMIB) bound at the Qp site also creates the SQ-
2Fe2S state with a g = 1.94 transition. However, in this case the state was already observed under equilibrium conditions, without PC present. In fact, its stability is so high that it inhibits the Qp site. Indeed, the inhibition of Cytb6f by DBMIB has long been known, as has its effect on the shape of the EPR spectrum in the region of 2Fe2S, 84,108,483 This has been interpreted over the years as a structural alteration of the cluster caused by inhibitor binding 110. The frequency-dependent shift in the g value of the signal induced by DBMIB (similar to shifts discussed above) dismisses this interpretation, indicating that the signal must reflect a spin-spin exchange interaction between the SQ form of DBMIB and the reduced 2Fe2S 482 (see also section 5).

Considering all the details of the formation and properties of SQ-2Fe2S uncovered to date, an energy diagram was proposed for both Cytbc1 and Cytb6f, in which this state represents a local energetic minimum, placed below the state with the reduced heme bL/bp (given that SQ-2Fe2S is observed along with oxidized heme bL/bp), but above the state with reduced heme bH/bn (Fig. 32). It has been suggested that SQ-2Fe2S is a metastable state, which may play a role as a buffer for electrons, in particular under conditions when electrons cannot freely leave the low-potential chain through the Qi/Qn site. Although the physiological role of this state is not yet known, in view of the observed negative correlation between its presence and superoxide production, and the apparent non-reactivity of this state with molecular oxygen, it was proposed that SQ-2Fe2S may be associated with protection against side reactions at the Qo/Qp site. This concept further assumes that the probability of formation of SQ-2Fe2S might be adjusted to the oxygen tension in the cellular environment and varies between different species. This probability depends on the energy difference between SQ-2Fe2S and the states determined by the Em values of Q/QH2 couple and the Em values of the oxidized/reduced couples of low-potential cofactors of Cyt-bc (Fig. 32). This notion was proposed in view of the observation that Cytb6f, which experiences more than an order of magnitude higher levels of oxygen in chloroplasts than does Cytbc1 in mitochondria, appears to have a greater tendency to reside in this buffered state.

An intriguing hypothesis was put forward for a possible role of the Cytb6f SQ-2Fe2S in regulation of the electron transfer pathways in oxygenic photosynthesis. It was proposed that the SQ-2Fe2S state in Cytb6f might serve as a factor that changes the efficiency of CET vs LET between photosystem I and II. This concept was based on the assumption that transient stabilization of the SQ-2Fe2S temporarily blocks the oxidation of further PQH2 in the Qp site, thus creating conditions in which the probability of delivering the second electron needed to complete the reduction of PQ in the Qn site by Fd/FNR is significantly increased.482
<figure><img src="image_36.png" /></figure>

Figure 31. Two forms of SQ detected by EPR for the Q<sub>o</sub>/Q<sub>p</sub> site of Cytbc: the spin coupled SQ-2Fe2S state (with prominent transition at g= 1.945) and the “free” SQ<sub>o</sub> not coupled to 2Fe2S (with transition at g = 2.006). Experimental spectra of Cytbc<sub>1</sub> (A,C,D) and Cytb<sub>6f</sub> (B) recorded at the X band. Spectra in A and B contain contributions from the spectrum of SQ-2Fe2S and the spectrum of 2Fe2S. C shows the spectrum of 2Fe2 alone, for comparison. The insert (D) presents the single-line spectrum of the SQ<sub>o</sub>.

Figure 32. A simplified energy diagram of Cytbc₁ and b₆f. The diagram shows the states of the catalytic reaction at the Q₀/Qₚ site. 1) Conditions before the start of the bifurcation reaction - Q₀/Qₚ is occupied by QH₂, 2Fe2S and heme bL/bp are oxidized. 2) Transition state - unstable semiquinone and the reduced 2Fe2S, oxidized heme bL/bp. 3) Conditions after full oxidation of QH₂ - Q₀/Qₚ is occupied by Q, cluster 2Fe2S and heme bL/bp are reduced. 4) Stage after electron transfer from heme bL/bp to heme bH/bn. This state has different energy in the Cytbc₁ and Cytb₆f due to the different potential of heme bH/bn in both enzymes and the potential differences between PQ and UQ. 5) Spin-coupled SQ-2Fe2S state with
<figure><img src="image_37.png" /></figure>

oxidized heme $b_L/b_p$. The redox state of heme $b_H$ in all states (1-5) is not considered in the context of the system's energy. The level of the reaction of SQ with oxygen is also indicated as a gray line below state 2. ⁴⁸²

# 8 Mechanistic insights into the catalytic Qᵢ/Qₙ site

## 8.1 Overview of the structure of Qᵢ and the proton paths

The Qᵢ site binding pocket is located in the vicinity to heme $b_H$ near the *n* side of the membrane and its entrance opens towards the membrane core allowing substrate and product molecule to enter and leave the site. The pocket is surrounded by residues from transmembrane helices A, D, E, the amphipatic surface helix a, the DE loop and the N-terminal peptide. The crystal structures identified not only the position of site-specific inhibitor (antimycin A) ⁷⁹,¹²³, but also the position of bound substrate (UQ) ⁶¹,⁶³,⁷⁹,¹²³,⁶⁷³. The binding sites of these compounds were found to significantly overlap with little rearrangements in the protein backbone and side-chains, as compared with the rearrangement of the protein around the Q₀ site upon binding inhibitors. This indicates that the Qᵢ site is structurally more rigid than the Q₀ site.

The quinone ring bound at the Qᵢ site is nearly perpendicular to the plane of heme $b_H$ with the closest distance of 4.2 Å ⁷⁹. It appears to be stabilized by both hydrophobic and polar interactions. The former include interaction with $^tPhe220/S^cMet221$, the latter through formation of hydrogen bonds with three residues: $^tSer205/S^cSer206$, $^tAsp228/S^cAsp229$, and $^tHis201$ ⁷⁹,⁶⁷³. The His interacts directly with quinone in bovine ⁷⁹ and also in bacterial enzyme (His217) ⁶³, while in yeast ($^cHis202$)– it interacts indirectly through a water molecule.⁶⁷³

As the Asp and His residues interact respectively with O1 and O4 of Q, they are considered to be involved in protonation of Q. In addition to these residues, the crystal structures identified other
structural elements that could potentially form proton paths from the protein surface towards the O1 and O4 of Q.

The path toward the O1 (referred to as the D/K path) involves a conserved Lys residue and a bound cardiolipin, which interacts further with a conserved Asp located next to the bound Q (Fig. 33). The cardiolipin has been suggested to act as a proton attracting antenna, which transfers protons to the nearby Lys⁶⁷³. The Lys further passes the protons to the conserved Asp through a string of interconnected water molecules ⁶⁷⁴, or alternatively undergoes a rotation towards the Qᵢ site and directly interacts with the Asp, as suggested by MD simulations ⁷⁹,⁶⁷⁵.

The path toward the O4 involves a conserved Glu and a conserved Arg that may interact through the water molecules with a conserved His located next to the bound Q ⁶⁷³,⁶⁷⁴. Site-directed mutations of this residue fully inactivated bacterial Cytbc₁ ⁶⁷⁶. On the other hand the Asp/Lys path was found to be disabled only when both protonable groups were removed. With just one protonable residue from the Asp/Lys pair, the entrance of protons to the catalytic site was sustained, albeit at lower rates, indicating that protons in the Asp/Lys path can travel through parallel routes, possibly involving water molecules ⁶⁷⁷. The Asp/Lys path thus appears to tolerate disruption, as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site. In this context, the Qᵢ site is similar to other Q binding sites, such as the Q_B site of photosynthetic reaction center, for which a multiplicity for proton paths has also been considered ⁶⁷⁸

<figure><img src="image_38.png" /></figure>

Figure 33. Structural details of the region around the Qᵢ site. A) Overall view of the Q binding environment and elements involved in proton transfer to Q. B) Geometry of the Q binding at the Qᵢ site. Labels indicate O1, O4, C1 and C4 atoms of the Q molecule and distances between Q and proton donors. Models based on PDB IDs: A206 and 1NTZ, A and B, respectively.
## 8.2 Catalytic electron and proton transfers at the Qᵢ site of Cytc**<sub>1</sub>**

The reduction of Q to QH₂ at the Qᵢ site involves two sequential single electron transfer steps in which the electrons are delivered from the same cofactor chain ³⁹. Because the arrival of the electrons is separated in time, the reaction must involve the formation of an SQ intermediate (SQᵢ) that is stabilized within the Qᵢ binding pocket before the final product (QH₂) is formed and leaves the site. Indeed, a stable radical signal originating from the Qᵢ site was recognized in early EPR studies on submitochondrial particles ⁴⁴⁰ and crude protein extracts ¹⁴¹,⁶⁷⁹ and has been explored since then as a key mechanistic element of models proposed for that site ¹⁵¹,¹⁶³,²⁴⁵,⁶⁴⁷,⁶⁴⁸,⁶⁷⁷,⁶⁸⁰⁻⁶⁸⁴.

Considering electron transfer, reduced heme bH acts as immediate electron donor to Q and then to SQ formed after the first electron transfer (SQᵢ). These reactions are favored by the generally lower E_m of the heme compared to the average E_m of the Q(H₂) pool (under physiological pH, E_m7 of heme bH is around +50 mV, while that of the pool is around +100 mV) ¹⁴⁹. As these reactions leave heme bH in an oxidized form, the SQᵢ associated with the oxidized heme bH (SQᵢ-bH³⁺) is one of the key intermediate states of the catalytic forward reaction ².

At alkaline pH, the E_m of heme bH approaches that of the Q(H₂) pool and a partial reversal of the reaction can be observed. In this case QH₂ bound at the Qᵢ site reduces heme bH, resulting in formation of SQᵢ at this site ¹⁶³. As in this reaction, QH₂ instead of Q acts as a substrate for the Qᵢ site, and this is favored not only at alkaline pH but also at high QH₂ to Q ratios ⁶⁴⁷,⁶⁸⁵. This reaction, unlike the forward reaction, leads to the formation of SQ that is present along with reduced heme bH (SQᵢ-bH²⁺). For many years, SQᵢ-bH²⁺ has been the dominant form of SQᵢ detected and explored experimentally. However, more recent studies indicated that the analysis of both forms of SQᵢ (i.e. SQᵢ -bH³⁺ and SQᵢ -bH²⁺) is crucial to advance our understanding of the molecular mechanism of the operation of the Qᵢ site (see section 8.3).

In addition to the observation of a stable SQᵢ radical by EPR, the stability of SQᵢ is supported by data from light-induced electron transfer measurements. Under conditions of single-turnover at the Q₀ site, the offset in the levels of light-induced heme bH reduction observed in the absence and presence of antimycin corresponds to the amount of SQᵢ that is formed in the forward reaction (electron transfer from heme bH to Q). On the other hand, when the Q₀ site is blocked by an inhibitor, the level of light-induced heme bH reduction corresponds to the amount of SQᵢ formed in the reverse reaction (electron transfer from QH₂ to heme bH). The extent of the forward reaction reflects the difference between the E_m of heme bH³⁺/bH²⁺ and the E_m of Q/SQ couples, while the extent of the reverse reaction indicates the difference between the E_m of heme bH³⁺/bH²⁺ and the E_m of SQ/QH₂ couples. Further analysis of such extents might provide estimates of E_m for Q/SQ and E_m for SQ/QH₂, which in turn would provide information on the stability constant of SQ (log(Kₛ) = [E_m(Q/SQ) –
$E_m(\text{SQ/QH}_2)]/60$. Using this approach with a specific mutant in which the $E_{m,7}$ of heme $b_H$ was elevated to the level of the $E_m$ of the QH$_2$ pool, the stability constant of SQ$_i$ was estimated to be at the level of $3 \times 10^{-1}$.¹⁴⁹

## 8.3 Fast- and slow-relaxing SQᵢ as dominant intermediates of forward and reverse reactions at the Qᵢ site, respectively.

Until recently, EPR-based investigation of the SQᵢ intermediate relied on the assumption that the SQᵢ interacts antiferromagnetically with the oxidized heme $b_H$, forming an “EPR-silent exchange couple”. Therefore only SQᵢ present when heme $b_H$ is also reduced (SQᵢ -$b_H$<sup>2+</sup>) was thought to be spectroscopically detectable ¹⁴¹,⁶⁸¹. This notion is no longer valid in light of the recent studies that identified two distinct populations of SQᵢ differing in the rates of spin-lattice relaxation (Fig. 34 A) ¹⁵¹,¹⁶³. The first population, characterized by a slow relaxation (SQᵢ<sub>S</sub>), corresponds to the well-known and extensively examined SQᵢ -$b_H$<sup>2+</sup> state, whereas the second population, characterized by a significantly faster relaxation time (SQᵢ<sub>F</sub>), corresponds to the long-missing SQᵢ -$b_H$<sup>3+</sup> state. The dipolar interaction between SQᵢ<sub>F</sub> and oxidized heme $b_H$ manifests itself in the characteristic properties of SQᵢ<sub>F</sub>, including fast spin-lattice relaxation leading to dominant homogeneous line broadening ⁶⁸⁶ and unusual behavior of the signal amplitude that does not follow Curie's law ⁶⁸⁷. These findings established that, contrary to long-standing assumptions, the SQᵢ-$b_H$<sup>3+</sup> state is not antiferromagnetic and can be separated spectroscopically (as SQᵢ<sub>F</sub>) if an appropriate combination of microwave power and temperature is applied during EPR measurements ¹⁵¹
<figure><img src="image_39.png" /></figure>

Figure 34. A) Two populations of SQᵢ detected by EPR. Upper: slow-relaxing SQᵢ (SQᵢS) associated with the presence of reduced (and diamagnetic) heme b<sub>H</sub> near the Qᵢ site. Green line illustrates the expected MP saturation profile of SQᵢS. Lower: fast-relaxing SQᵢ (SQᵢF) associated with the presence of oxidized (and paramagnetic) heme b<sub>H</sub> near the Qᵢ site. Blue line illustrates the expected saturation profile of SQᵢF. B) EPR-monitored redox titrations of SQᵢS and SQᵢF in a range of pH from 5 to 9.<sup>163</sup>

Further studies revealed that SQᵢS and SQᵢF are separable in equilibrium redox titrations <sup>163</sup>. Remarkably, over a pH range from 6 to 8, SQᵢF represented a large, if not dominant, portion of the whole population of titrated SQᵢ, indicating that early titrations <sup>245</sup> had indeed missed a significant
fraction of SQ in this pH range (Fig. 34 B). SQᵢF was also trapped as a stable intermediate at the Qᵢ site during steady-state turnover <sup>163</sup>, which is consistent with the electron transfer reaction scheme predicting SQᵢ⁻bH<sup>3+</sup> as the key intermediate state of the catalytic forward reaction (heme bH to Q electron transfer) <sup>2</sup>. On the other hand, SQᵢS appeared as the dominant intermediate state observed under the conditions favoring the reverse reaction (QH<sub>2</sub> to heme bH electron transfer).

Interestingly, when a single electron gets injected into heme bH of one monomer through the reverse reaction at the Qi site, there is a possibility of electron equilibration within all four hemes of the b-chain of the dimer (through the heme bL-bL bridge, see figure 5 and section 9). This way the electron can reach the second Qi site (reducing Q to SQi) which overall will result in a formation of SQIF in both monomers (Fig. 35). The observation that both SQis and SQiF are observed in the native enzyme with an inactivated Qo site (when only the reverse reaction at Qi is possible) indicates that electron equilibration within the b-chain of the dimer indeed traps some fraction of the molecules of Cytbc₁ with the SQi⁻bH<sup>3+</sup> state at both Qi sites <sup>151</sup>. This conclusion was further supported by the observation that electron redistribution over b hemes /Qi sites taking place within the dimer and monitored by analyzing the change in relative proportions of SQiF / SQis, is effectively modulated in mutants that significantly raise the redox midpoint potential of either heme bL or heme bH. <sup>149,151</sup>

<figure><img src="image_40.png" /><figcaption>Figure 35. The cross-dimer electron equilibration leading to formation of SQᵢF (see details in the text)..</figcaption></figure>

**8.4 Charge polarization of SQᵢ facilitates electron and proton reactions at the Qᵢ site.**

The experimental exploration of both states of SQᵢ (i.e. SQᵢS and SQᵢF) complemented by
quantum mechanical calculations lead to the formulation of a hypothetical mechanism which places
importance on the charge and spin polarization of SQᵢ intermediates in the catalytic reactions at the
Qᵢ site ¹⁶³. This polarization is imposed by the local electrostatic field induced by the specific
distribution of residue charges within the Q-binding pocket. These are mainly His in the vicinity of
the O4 atom and the Asp/Lys pair in the vicinity of the O1 atom.

This “charge polarization” mechanism is schematically presented in Fig. 35, right. The
evolution of states A to G considers the direction of reduction of Q to QH₂. The reaction requires
initial entry of Q to the Qᵢ site (states A and B) and heme bH in reduced state (state C). It starts when the O4 atom of the Q present at the site interacts with the protonated His. The electron is then transferred from this heme to Q resulting in the formation of the SQᵢ anion (which is detected by EPR as SQIF when heme bH is oxidized) (state D). The anionic SQᵢ becomes more negative at the O4 atom than at the O1 atom, while the spin density is larger at the C1 side compared to the C4 side (Fig. 35, left). Such polarization and the resulting geometry favor a barrierless exchange of the proton originating from His between the His imidazole ring and the O4 atom of SQᵢ. In this state, the SQᵢ radical is stabilized, and equilibrates between the anionic and neutral forms. This equilibration also takes place after the reduction of heme bH and is associated with the conversion of SQIF to SQIS (conversion of state E to F). This continues until the second electron reduces the neutral SQᵢ to unstable QH⁻ (state F). When this equilibration takes place, the protonation of anionic SQᵢ to the neutral form does not influence the spin density but causes an inversion of the direction of charge polarization: the C1 side becomes more negative than the C4 side. This increases the probability that the second proton will be transferred to the O1 atom. Consequently, the reduction of neutral SQᵢ to QH⁻ is accompanied by the spontaneous transfer of a second proton from the Asp/Lys-path to the O1 atom, completing the reduction of Q to QH₂ followed by dissociation of the product (state G).

<figure><img src="image_41.png" /></figure>

Figure.36. The charge polarization mechanisms of Q reduction at the Qᵢ site. Boxes on the right represent the reaction steps A to G (see text for details). SQᵢ bound at the Qᵢ site (blue and green boxes refer to the Qᵢ site occupied by SQIF and SQIS, respectively) is stabilized by equilibration between neutral and anionic form of the radical (upper and lower scheme
in the left panel, respectively). Red and blue circles denote the negative and positive Mulliken charges, respectively.
Radius of each circle is proportional to the charge of the respective atom (the values are shown as black numbers). Spikes
on the left indicate the polarization of overall charge on the respective SQ form.¹⁶³

The key feature of the proposed mechanism is an initial response of the radical anion to the
local electrostatic field, as it triggers progression of the whole catalytic reaction. At the same time,
polarized SQᵢ exchanging a proton in the absence of barrier with His forms a stable intermediate
which, during enzymatic turnover, safely “waits” for the second electron without the risk of
generating other radicals (such as superoxide).

The charge polarization mechanism sheds new light on the protonation states of SQᵢ described earlier by the pulse spin-echo-based EPR (ESEEM or HYSCORE)⁶⁴⁷,⁶⁴⁸,⁶⁸³, which must have referred to just the SQᵢS, as the spin-lattice relaxation of SQᵢF is fast enough to lead to homogeneous line broadening that precludes the formation of a spin-echo signal. It was proposed that SQᵢ (SQᵢS in view of the above assumption) is likely to be anionic ⁶⁴⁸,⁶⁸² and ligated by a hydrogen bond from the imidazole-ring nitrogen of the His ⁶⁴⁷,⁶⁴⁸. It was also suggested that the redox state of heme bH might influence the protonation state of SQᵢ and the His ligand, rendering SQᵢ deprotonated at the time when heme is reduced ⁶⁸³. This charge polarization mechanism implicates that SQᵢ exists as neither an anion nor a neutral radical but should rather be treated as a resonant structure in which the proton undergoes exchange between His and SQᵢ. If the frequency of this exchange is much higher than the hyperfine splitting of the radical spectrum caused by the interactions of the proton with unpaired electron spin, then EPR will be unable to detect the presence of protons due to large spectral diffusion effects.

The proposed catalytic role of charge and spin polarization of the SQ is likely to be relevant
to other sites catalyzing the reduction/oxidation of Q/QH₂ via stable SQ intermediates. Such spin
polarization has been noted before for the SQ bound at catalytic sites (including SQᵢ⁶⁸⁰, SQH of quinol
bo₃ oxidase ⁶⁸⁸ and SQA of bacterial reaction center ⁶⁸⁹), and could be associated with charge
polarization induced by the electrostatic field provided by the catalytic site. Further support for this
proposal comes from modeling of the effect of the electrostatic field on spin and charge polarization
in diboryl monoradical anions ⁶⁹⁰.

**8.5 Specific residues involved in PQ/PQH₂ binding to the Qn site of Cytb6f**
Several attempts have been made in the past to study residues important for Q/QH₂ binding to the Qn site of Cytb₆f via site directed mutagenesis approaches informed by previous results from work on the Cytbc₁ complex and by structural features of the Cytb₆f. They include residues bG37 and bF41,⁶⁹¹ because substitutions of the corresponding residues in *Rhodobacter*'s Cytb (G48 and A52 respectively) led to impaired redox activity, loss of photosynthetic growth or failure to assemble a functional complex. The F41V mutant of *Chlamydomonas* displayed a 5-fold decrease in Qn site activity and became sensitive to antimycin, an inhibitor of the Cytbc₁ Qᵢ site that is normally not effective against Cytb₆f. Conversely, the bF41G and bF44A mutants did not display any phenotype⁶⁹². Substitutions of the Gly37 residue did not rescue phototrophic growth, suggesting that mutation of this residue is detrimental to photosynthesis. The same result was found in the case of a C35V mutant, which was unable to grow phototrophically because this substitution (affecting the residue that covalently bonds heme cₙ to the Cytb₆) prevents the accumulation of the entire complex in thylakoids.⁶⁹³

Other mutants were generated affecting residues in the proximity of the heme: Leu36, Pro41 of SubIV. No clones were recovered after mutagenesis, indicating that these mutations were also detrimental for photosynthesis. On the other hand, a mutant was obtained after the P40T substitution. This mutant showed a significant slowing down of Cytb₆ reoxidation under single turnover flash illumination, consistent with impairment of interaction between the heme and its quinone substrate. The mutant also displayed a downshift of the cₙ heme midpoint potential (from +100 mV to -200 mV at pH 7), but conserved WT Cytb₆f turnover rates under a continuous light regime. Based on the position of P40 in the Cytb₆f structure, it was proposed that this modified accessibility could originate from the ligation of the heme iron by the phenol(ate) side chain introduced by the mutation ⁴³⁰.

A similar approach has been used in cyanobacteria, where a mutant (R214H) was generated to substitute His for a conserved Arg in the Cytb₆ polypeptide of the cyanobacterium *Synechococcus* sp. SPCC 7002. At high light intensity, the R214H mutant grew ~2.5-fold more slowly than the WT, due to inhibition of the Cytb₆f complex turnover. Under single turnover flash illumination the R214H mutation partially blocked electron transfer to the Qn site, mimicking the effect of the Qn site inhibitor NQNO. Overall, these data define Pro 40 and Arg 214 as key residues for catalysis in the Qn site of the Cytb₆f⁶⁹⁴.

Other mutants were generated to modify the environment of the chlorophyll ring that is located close to the Qn site⁶⁹⁵. Residues Val104, Gly136, Tyr124, and Arg125 did not dramatically affect electron transfer in the complex, but strongly affected state transitions (see section 10.3 below).
This indicates that besides catalysis, the reduction of the PQ at the stromal side Qn site is also involved in sensing and signaling, two essential functions of the Cytb6f (see sections 5.2 and 10.3)⁶⁹⁶

## 9 Inter-monomer electron transfer

Crystallographic structures of Cytbc1 and Cytb6f revealed a high degree of symmetry between monomers including structural arrangement of the redox active cofactors²⁰⁻²²,⁴⁸,¹⁷³. One of the most intriguing structural features of the dimer is the distance between hemes $b_L/b_p$ of different monomers, which theoretically seems to be small enough to allow fast (i.e. ~μsec) electron transfer ⁵⁶,¹⁹⁵,⁶⁹⁷,⁶⁹⁸, in relation to catalytic rates ⁴¹,⁶⁸,⁶¹⁵,⁶⁹⁹. For this reason, a discussion began about the possible implications of such an electronic connection between monomers for catalysis and opened the door to construction of models extensively utilizing the heme $b_L-b_L$ bridge ⁸⁶⁻⁸⁸,¹²⁵,³²⁹,⁷⁰⁰,⁷⁰¹.

The fundamental question therefore arises as to whether electron transfer between the $b_L/b_p$ hemes of the two monomers can be considered competitive to the intra-monomer path: from heme $b_L/b_p$, through heme $b_H/b_n$ to the Qi/Qn site. Alternatively, is this simply a redundancy of the possible electron transfer paths that may be important only under specific and rare conditions²⁰⁶,⁵⁷¹,⁶¹⁵.

The order of the rates for inter-monomer electron transfer can be approximated on the grounds of electron transfer theory ⁷⁰² and the phenomenological approximation of the Moser-Dutton ruler ⁵⁶,¹⁹⁵,⁶⁹⁷,⁶⁹⁸:

$$log(k_{ET}) = 13 - (1.2 - 0.8\rho)(R - 3.6) - 3.1 \frac{(\Delta G + \lambda)^2}{\lambda},$$

where: $\rho$ – is packing density (0 for vacuum, 1 – fully packed matrix), $\Delta G$ – Gibbs energy associated with electron transfer from donor to acceptor (in the case of electron transfer from heme $b_L$ to $b_L$ this is 0) and $\lambda$ – reorganization energy, $R$ – distance between donor and acceptor (between $b_L$ heme-rings)

Using average values for electron transfer within a protein matrix – $\rho$ and $\lambda$ in the above equation can be simplified to account only for $R$⁶⁹⁷:

$$log(k_{ET}) = 12.8 - 0.6R$$

As distances between heme rings are ~13.2 – 15.2 Å, a crude estimation of the rates gives $k_{ET}$ ~ 5×10³ – 8×10⁴ s⁻¹. A typical steady-state turnover rate obtained for Cytbc1 or Cytb6f does not exceed
$10^3 s^{-1} 569,649$ meaning that electron transfer between hemes $b_L/b_p$ should not limit the catalytic turnover rate.

The experimental verification of whether intermonomer electron transfer is indeed as fast as theory predicts, required measuring the electron transfer rate between $b_L/b_p$ hemes in the dimer, which in the native protein is generally difficult considering the structural symmetry of the complex, and the fact that electron transfer between the monomers leads to spectroscopically indistinguishable states. In fact, this has long been out of reach for direct experimental testing, due to the lack of a genetic tool to inactivate individual components of the dimer in an asymmetric manner. This is related to the fact that, in native systems, the genes coding for the subunits do not differentiate between monomers, thus any mutation is obligatorily introduced to both parts of the homodimer.

New strategies described in 2010 aimed at overcome these limitations.<sup>47,329,703–707</sup> In these studies two distinct genetic approaches were used: a fusion duplicating gene or a co-expression of two variants of the gene. Both targeted the Cytb subunit of bacterial Cytbc<sub>1</sub>.

The first approach was based on extending the native gene encoding Cytb with a short linker followed by a second copy of the same gene<sup>47,705–707</sup>. As a result, each half of the fusion protein replaced one Cytb subunit of the dimer, providing a template for construction of derivatives in which the two halves could be mutated independently. Using such a template, different mutations inactivating key steps of electron transfer were introduced in various combinations to eliminate selected paths and to force electron transfer through other available paths within the dimer (see all forms in Fig. 37). This was used to test all possible electron transfer routes for electrons from the $Q_o$ to $Q_i$ site.<sup>47,707</sup>

Because for a given combination of mutations, only one version of the complex with the fused Cytb is expressed in the cells, it was possible to monitor the kinetics of light-induced electron transfer in the native membranes. Analysis of the cross-inactivated complex (a variant wB-B<sup>N</sup> in Fig. 37), for which electrons derived from the $Q_o$ site must have crossed the dimer to reach the $Q_i$ site, revealed that electron transfer between hemes $b_L$ occurs on a millisecond time scale, as theory had predicted. In addition, it was established that the complex remains enzymatically active even if the equivalent of only one monomer (w<sup>N</sup>B-B), or the equivalent of one monomer with a part of other monomer (wB-B, <sup>N</sup>B-B) are available. This revealed that any path connecting either or both $Q_o$ sites with either or both $Q_i$ sites is competent in supporting enzymatic turnover at high rates<sup>47,704,705</sup>. Based on these findings it was proposed that cofactor chains in the native dimer assemble into an H-shaped electron
transfer system (two upper, two lower branches and a bridge) connecting the two Q₀ sites with the
two Qᵢ sites.

Interestingly, deactivation of only one branch (forms wB-B, N'B-B) or both branches of the
same monomer (wN'B-B) decreases Vmax of the enzyme by approximately 50% when compared to the
fusion protein without any disabling mutation (B-B) ⁵⁶. This suggests that the two monomers in the
dimer contribute equally and independently of each other, thus any allosteric effect ¹⁹,⁸⁷,⁸⁸,⁷⁰⁸, if it
exists, does not govern the catalytic turnover rate. Forcing electron transfer through the bL heme
“bridge” decreases Vmax to ~70 s⁻¹, which is about 25–40% of the average activity obtained for wB-
B, wN'B-B and N'B-B forms, but is still enough to support multiple turnovers at high rates.

When using a fusion gene composed of two copies of the same gene, careful procedures must
be employed in order to maintain high expression of the fusion protein during cell growth and prevent
gene recombination.⁷⁰⁶ Without rigorous growth and harvesting protocols there is a risk of losing the
fusion or asymmetry of the mutations, which is especially critical if one wishes to test Cytbc₁-related
support of photosynthetic growth of bacterial cells. Indeed, just such recombination was observed,
but incorrectly interpreted by Hong et al. ⁷⁰⁹

To overcome these limitations and gain the possibility to test functionality of the
asymmetrically mutated complexes *in vivo*, the original system was modified by constructing a hybrid
fusion system, in which the fusion protein was expressed from a gene linking two different genes
encoding *Cytb* from closely related bacteria *Rhodobacter sphaeroides* and *capsulatus* ⁷⁰⁴,⁷⁰⁵. Careful
analysis of photosynthetic competence of the various complexes containing asymmetrically mutated
hybrid fusion proteins (analogous to the ones described above) demonstrated that in agreement with
kinetic results, the cross-inactivated form relying exclusively on inter-monomer electron transfer is
fully functional *in vivo*, as is the form with just one branch inactivated.
<figure><img src="image_42.png" /></figure>

Figure 37. Schematic representation of various forms of *Cytbc*₁-like complexes containing *Cytb* fusion protein used to test possible combinations of electron transfer paths. A) Native-like system with all four branches active (B-B). B-E) Asymmetric forms containing one or more mutations inactivating either the Q₀ site (*Cytb*:RhG158W; designated as wB) or heme *b*H (*Cytb*:RhH212N; designated as BN): wB-B – lower branch inactivated, NB-B – one upper branch inactivated; wNB-B – upper and lower branch on the same side inactivated; wB-BN – cross-inactivated enzyme.

A second approach for obtaining asymmetrically mutated variants of *Cytbc*₁ was based on co-expression of two plasmids, each carrying one copy of the gene encoding for *Cytb* with a sequence coding for a different tag attached (His-tag, Flag-tag or Strep-tag) ³²⁹,⁷¹⁰,⁷¹¹. The different mutations inactivating either the Q₀ or the Qᵢ sites were placed on different plasmids and the two-step affinity chromatography was used to isolate the cross-inactivated form from the symmetric variants resulting from random assembly of the complexes in the cells ⁷⁰³,⁷¹⁰. These studies also demonstrated that inter-monomer electron transfer through *b*<sub>L</sub> hemes occurs in milliseconds, thus in a catalytically-relevant timescale. In addition, some of the kinetic transients were interpreted as resulting from electron equilibration between the two Qᵢ sites and the four hemes *b* in the dimer ⁷⁰³. The same type of equilibration (see Fig. 35) was considered as rationale for the existence of fast-relaxing semiquinone
(SQᵢF) in native Cytbc₁ exposed to conditions favoring one electron reduction of heme bH by QH₂ bound at the Qᵢ site (described in section 8.3) <sup>151</sup>.

Although the existence of inter-monomer transfer has been confirmed by several studies, its
possible physiological meaning and role in the catalysis remain unclear.<sup>13,699</sup> Electron transfer
between the bL hemes could be just a simple coincidence of the conserved structure of the homodimer
and as such does not play an important catalytic role, as all structural elements needed to perform
catalytic cycle are present in each of the monomers<sup>23,206,709</sup>.

On the other hand, it has been hypothesized that inter-monomer electron transfer to support
efficient catalysis may offer some advantage for bioenergetic processes, in particular when
unfavorable redox conditions and/or potential danger of superoxide generation exist. As pointed out
by Sarewicz et al.<sup>13,699</sup> mitochondria usually posses several copies of genes coding for Cytb and the
appearance of a mutation in one or more genes is expected to lead to expression of a fraction of the
dimers having damage in only one part of the monomer (and thus possibly having a “dead end” for
electrons). If this monomer was isolated functionally from undamaged monomer, it would inevitably
catalyze only energy-wasting short-circuits or free-radical generating reactions. However, if the
damaged monomer is linked functionally with an undamaged one, the existence of the heme bL-bL
bridge can provide an efficient way to remove electrons from the low-potential chain in the damaged
monomer (Fig. 38). With such a mechanism, the amount of ROS generated during the QH₂ oxidation
will be significantly lowered enabling electron flow through respiratory or photosynthetic chains.

Also, as shown by Pintscher et al.¹⁵¹ electron exchange between monomers may increase the rate of SQᵢ reduction to QH₂ by providing electron paths for dismutation of SQ⁻ from two Qᵢ sites of a dimer, which can be of advantage under shortage of QH₂.

<figure><img src="image_43.png" /></figure>

Figure 38. Putative advantage of dimers with possible ET through the hemes bL. A) Homodimeric Cytbc₁ operates normally using two active monomers. B) A heterodimer in which one monomer has disabled heme bH and or Qᵢ site (gray) is still still functional and able to oxidize QH₂ at two Q₀ sites. C) A heterodimer containing two different mutations in each monomer, one with disabled Q₀ site (green) and second with heme bH or Qᵢ site, still catalyze QH₂ oxidation. D) and E) Homodimers containing mutations are inactive and/or contribute to elevated superoxide generation. Considering a sum of monomers from B to E, occurrence of intermonomer electron transfer gives: 3 active monomers, 3 inactive monomers
and 2 monomers generating O₂⁻. If this electron transfer did not exist we would have 1 active monomer Q₀ sites, 3 inactive monomers and 4 monomers generating O₂⁻.

There are several models presenting more or less elaborate mechanisms of inter-monomer allosteric communication in which the role of heme $b_L-b_L$ electron transfer is crucial for catalysis. Most of these were inferred from indirect kinetic measurements and/or effects of substoichiometric additions of inhibitors. The results of such experiments leave space open for discussion and future experimental exploration. While the existence and potential significance of allosteric effects have never been ruled out, the experiments that exploit the family of fusion proteins suggest the simplest mechanism, in which electrons travel "blindly" using any available path through the H-shaped electron transfer system to complete redox reactions without generation of radicals.

# 10 Higher level of organization and regulation (supercomplexes, state transition, kinase activation)

## 10.1 A role for cytochrome $b_6f$ in the regulation of linear vs cyclic electron flow?

Alternative to linear electron transfer (LET), which requires in series PSII and PSI activity, is the cyclic pathway (CET), which involves electron flow via PSI and the P700 reaction center complex via the plastoquinone pool through Cytb₆f complex. It thus produces ATP without generating reducing power, and it is considered as a central element to readjust the ATP/NADPH balance for proper CO₂ assimilation in plants and green algae <sup>230</sup>. Actors involved in CET and CET regulation have been recapitulated in recent reviews <sup>374,712-715</sup>. Nonetheless, several aspects of this process remain enigmatic. While most of the CET electron carriers (the plastoquinone pool, Cytb₆f, plastocyanin, PSI and ferredoxin and possibly NADPH) are shared with the LET pathway, electron transfer from Fd/NADPH to the quinone pool on the stromal side of the thylakoids may require an additional, CET specific, activity. This activity would be either by the NAD(P)H dehydrogenase (NDH) complex similar (but not identical) to Complex I of the mitochondrial respiratory chain <sup>713,716</sup>, or the FQR (ferredoxin quinone reductase) complex. FQR would correspond either to a PGRL1 and PGR5 complex <sup>199</sup> or to the Cytb₆f itself either Cytb₆f complex interacting with Fd, most likely via FNR (as discussed paragraph **错! Source du renvoi introuvable.**) and/or possibly PGRL1 and PGR5, although as considered above involvement of the latter components is supported only by very qualitative electron transfer data and stoichiometries are small.
Mutants lacking the NDH complex lack a clear growth phenotype under a variety of environmental conditions⁷¹⁶,⁷¹⁷, suggesting that the NDH activity, per se, is not essential for CET, since CET is an essential process for plant growth²³⁵. On the other hand, a mutant with enhanced CET activity, isolated in *Arabidopsis*, displays higher levels of the NDH complex³⁷⁹. Moreover, *Arabidopsis* photosynthetic embryos, which have a larger CET activity than mature leaves, also display an enhanced NDH activity⁷¹⁸. While the FQR activity was initially identified with the *Cytb6f*⁷¹⁹, this function has later been associated with a complex containing the PGR5, PGRL1 proteins¹⁹⁹, i.e. previously identified as CET actors⁷¹³. Similarly to the NDH, mutants lacking the PGR5:PGRL1 complex show WT like CET rates in *Arabidopsis* ³⁸⁶ and *Chlamydomonas* ³⁸⁷, again pointing to a non-essential role of this complex in CET.

Overall, these results can be interpreted assuming that both the NDH and FQR pathways contribute to CET, each activity being compensated by the other when genetically disrupted. Consistent with this conclusion, previous genetic studies have highlighted that severe growth phenotype in *Arabidopsis* can only be observed upon concomitant disruption of the NDH and PGR5:PGRL1 complexes⁷²⁰.

What is role for the *Cytb6f* (if any) in a context where the role of the NDH and PGRL1:PGR5 pathways in CET is still controversial? Interactions of the *Cytb6f* with PSI and/or FNR have been proposed as a mean to trigger CET ⁷²¹,⁷²². This notion has received experimental support by the finding by Cramer and co-workers that *Cytb6f* co-purified with FNR in plants ¹⁸⁰. However, recent ITC experiments show that there is no significant interaction between FNR and *Cytb6f*. Moreover, interactions between subunit of PSI, *Cytb*₆, FNR and PGR5 were reported in a yeast two-hybrid assay using *Arabidopsis* genes²³⁶. In the green alga *Chlamydomonas reinhardtii*, Minagawa and colleagues¹⁹⁷ were able to isolate a PSI:Cytb₆f, which they considered as a specific component of CET. The existence/function of this complex is still debated. If real, why would a supercomplex be needed for CET? As mentioned above, most electron carriers are shared between the CET and LET pathways. Therefore, CET, which is a ‘circular’ process continually recycling electrons around PSI, would be easily overcompeted by LET every time that a cyclic electron is shunted towards the stromal sinks by LET. Two alternative models have been proposed to explain how LET does not overcompete CET ⁴²⁶,⁷²³,⁷²⁴.

The first one considers that CET and LET are, to some extent, segregated ('segregated CET'). It is well-known that the photosynthetic complexes are unevenly distributed in the thylakoids: PSII is mainly concentrated in the appressed grana, PSI is mainly found in the non appressed membranes (stroma lamellae, grana margins), while the *Cytb6f* is roughly homogeneously distributed ⁷²⁵. Moreover, the diffusion of PQ/PQH₂ in the thylakoid membrane is highly restricted, probably due to
the very high protein concentrations that characterize this membrane<sup>272,726</sup>. The ‘segregated CET’ model assumes that because the physical distance between the different domains is rather large and the PQ diffusion in the thylakoids is restricted, two electron carrier pools would exist with different functions. Photosynthetic complexes present in the grana and grana margins (PSII, Cytb<sub>6f</sub> and PSI) would be involved in LET, while those in the stroma lamellae (Cytb<sub>6f</sub> and PSI) would be the CET ones. Consistent with this hypothesis, recent affinity-mapping AFM studies has revealed nanodomains of co-localised PSII and Cytb<sub>6f</sub> complexes<sup>727</sup>. The domains (previously hypothesized by Lavergne and Joliot<sup>272</sup>) would provide a structural rationale for the segregation of LET and CET and its regulation. Small changes in the distance between adjacent membranes in stacked grana regions might control the access of Cytb<sub>6f</sub> to the grana because of steric hindrance<sup>5</sup>. Because the width of the stromal gap is variable and light regulated, this model predicts that changes in the lateral distribution of the Cytb<sub>6f</sub> should affect CET and LET by modulating the amount of this complex present in the CET and LET compartments. Obviously, the presence of a supercomplex is an extreme case of the ‘segregated CET’ hypothesis, the CET domain becoming the PSI:Cytb<sub>6f</sub> supercomplex itself.

Alternative to the ‘segregated CET’ model, a ‘dynamic’ hypothesis to explain CET cyclic flow has been proposed<sup>410,426,723,728</sup>. According to this model, CET and LET would not be segregated but actually directly compete for electrons. Under most physiological conditions, this competition would favour LET, because of electron leak toward stromal sinks. However, under particular conditions (high light, CO<sub>2</sub> limitation, low oxygen in microalgae) CET would become more efficient because the LET efficiency would be reduced by limitation at the PSI acceptor side. Competition would occur either at the PSI acceptor side (via competition for Fd, likely via the FNR- Cytb<sub>6f</sub> complex<sup>723</sup> or at the level of the PQ(H<sub>2</sub>) pool<sup>374</sup>. The model proposed by these authors is particularly interesting from a Cytb<sub>6f</sub> perspective, as it postulates that a function interaction between the two Cytb<sub>6f</sub> monomers at the PQ-reducing site would allow a ‘direct’ electron flow from the stromal to the luminal PQ, in a confined environment not in contact with the photoactive PQ(H<sub>2</sub>) pool in the thylakoids, similar to the semiquinone version of the Q cycle proposed earlier<sup>729</sup>. This hypothesis is therefore somehow in between the ‘segregated CET’ and the redox competition hypotheses previously described.

## 10.2 The “elusive” Cytb<sub>6f</sub>-PSI CET supercomplex.

As discussed above, the presence of a PSI- Cytb<sub>6f</sub> complexes represent the most extreme case of the ‘segregated CET’ hypothesis. In the green alga *Chlamydomonas reinhardtii*, where CET can be a prominent process<sup>728,730,731</sup> Iwai and colleagues used a sucrose density gradient to isolate a supercomplex composed of the PSI-LHCI supercomplex with LHCIIs, Cytb<sub>6f</sub>, FNR, and PGRL1 in
a fraction heavier than that of the single complexes (PSI-LHCI, Cytb6f) alone. Its formation is enhanced by reducing conditions, which increases CET activity in this alga ²²¹,⁷³². Spectroscopic analyses ⁷³² indicate that PSI and Cytb6f are able to exchange electrons within the supercomplex, PSI being able to reduce Cytb6 in the presence of Fd and NADPH and to oxidise Cytf in the presence of plastocyanin ¹⁹⁷. Based on these results, the authors conclude that the PSI- Cytb6f supercomplex corresponds to the CET supercomplex, the existence of which had been anticipated by theoretical predictions ⁷³³ and functional studies (e.g. Joliot and Joliot ⁷³⁴).

Supercomplex formation in *Chlamydomonas reinhardtii* requires not only reducing conditions (anaerobiosis) but also Ca²⁺ signaling in *Chlamydomonas reinhardtii* ¹⁹⁷,⁷³⁵. Regulation by anaerobiosis would be mediated by a protein, Anaerobic Response 1 (ANR1). ANR1, as well as the plastid localized Ca²⁺ sensor (CAS) are found in the PSI- Cytb6f supercomplex, together with PGRL1 ⁷³⁵. The Cytb6f associated subunit PETO co-fractionates with other thylakoid proteins involved in CET (ANR1, PGRL1, FNR). The absence of PETO impairs the formation of the supercomplex in anaerobic conditions.

Since its discovery and biochemical characterization, the existence/function of the *Chlamydomonas* CET supercomplex has been debated. The biochemical significance of the green band of high molecular weight in sucrose density gradient that was used to purify this complex has been recently questioned, because various PSI complexes can migrate in these sucrose density regions ³⁷⁶. On the other hand, single-particle analysis of the PSI- Cytb6f complex from *Chlamydomonas* ³⁷⁶ has provided a structural support for the existence of a true supercomplexes in this alga.

Until recently, no such supercomplex was found in plants ⁴²⁶, and it was argued that no such supercomplex is required for CET to occur in plants ⁴¹⁰,⁷¹⁴. However, the isolation a PSI-Cytb6f supercomplex has been reported starting from stroma lamellae of *Arabidopsis* ⁷³⁷. However, even in this case the significance of this complex has been questioned ³⁷⁴, because in this preparation Cytb6f complex bound to PSI was not only in its active (dimeric) conformation but also in the inactive (monomeric) one.

At variance with the respiratory chain, where several supercomplexes between different component of the electron flow chain have been characterized, no other photosynthetic supercomplex has been reported so far, besides the PSI-NDH supercomplex that was characterized in *Arabidopsis* ⁷³⁸. Formation of the PSI-NDH supercomplex requires the presence of specific forms of the PSI Light Harvesting Complex (LHCI, ⁷³⁹).
10.3 Role of cytochrome *b*<sub>6</sub>*f* in state transitions

In oxygenic photosynthesis, the two photosystems have distinct pigment containing antenna complexes, and consequently different spectral absorption features. PSI has an enriched light absorption capacity in the far-red region, while PSII has a slightly higher absorption capacity in the spectral regions where chlorophyll *b* absorbs. Under natural light conditions, where both the quality and quantity fluctuate with time ⁷⁴⁰ light absorption by these two complexes may be unbalanced, with negative consequences on the quantum yield of photosynthesis, which requires a commensurate light utilization by PSI and PSII for optimum photosynthetic yield.

In plants and green algae, balanced light utilization is achieved via a regulatory mechanism called state transitions, which actively adjusts the absorption capacity of PSI and PSII, in response to chromatic changes in the light. Discovered by Murata and Sugahara (1969) and Bonaventura and Myers (1969) ⁷⁴¹,⁷⁴² state transitions rely on a redox triggered phosphorylation of the PSII antenna complexes (LHCII), which dissociates from PSII binding to PSI under conditions where absorption of the former is enhanced (e.g. when the light available for photosynthesis enhances light absorption by chlorophyll *b*). This state is called state 2. Conversely, over-excitation of PSI (e.g. by far red light) leads to the detachment of LHCII from PSI, and its re-association to PSII, leading to state 1, ²³⁰.

Pharmacological and genetic experiments have pinpointed the PSII electron acceptors (the
plastoquinone, PQ) pool as the sensor triggering LHCII phosphorylation ⁷⁴³. This occurs via the
activation of a specific kinase ³⁵, mediated by the Cytb6f. The plastoquinone can act as the redox
sensor because it is functionally 'located' between PSII and PSI. Thus, this redox active molecule
'senses' the relative imbalance in the light harvesting capacity of the two photosystems, becoming
reduced when the PSII activity overcomes that of PSI, and oxidised when PSI is prevailing. The
kinase responsible for LHCII phosphorylation has been first identified in Chlamydomonas, STT7 ⁷⁴⁴
and later in Arabidopsis, STN7 ⁷⁴⁵. It is as a ser/thr kinase located in the thylakoids. A phosphatase
(TAP38/PPH1) has also been found, which dephosphorylates LHCII ⁷⁴⁶,⁷⁴⁷ during the state 2 to state
1 transition.

Several pieces of evidence suggest that the PQ(H₂) pool activates the Stt7/STN7 kinase via Cytb₆f. Wollman and Lemaire ⁷⁴⁸ used Chamydomonas lines with mutations in the photosynthetic apparatus to demonstrate that state transitions were missing in mutants lacking the Cytb₆f. Later, independent spectroscopic ⁷⁴⁹ and genetic evidenced ⁵⁷⁶ suggested that the Cytb₆f mediates kinase activation upon PQH₂ binding to its lumenal binding pocket, via a still largely unknown mechanism.

Several models have been proposed so far to account for the Cytb₆f mediated activation of the sTT7/STN7 kinase ⁷²⁴,⁷⁵⁰⁻⁷⁵³. They are summarized in a multistep model proposed in Fig. 39. There, PQH₂ binding to the cytochrome lumenal pocket would induce conformational changes (via
movements of the ISP-HD) making the *Cytb*<sub>6f</sub>-kinase interaction possible. Formation of a *Cytb*<sub>6f</sub>-kinase complex would activate this protein leading to the phosphorylation of the *Cytb*<sub>6f</sub>-associated subunit PetO<sup>743,754,755</sup>. This step would precede LHCII phosphorylation, implying the existence of an additional step, where the active kinase would be released from *Cytb*<sub>6f</sub> to interact with the PSII antenna. The existence of two separate steps for *Cytb*<sub>6f</sub> and LHCII phosphorylation is suggested by the finding that PQ analogue stigmatellin, which blocks *Cytb*<sub>6f</sub> activity likely by interfering with the movement of the ISP-HD<sup>61,554,755</sup> prevents LHCII phosphorylation but not the phosphorylation of PetO.

This model also accounts for additional information concerning the induction of state transitions. The first one is how the signal generated by PQH₂ binding in the lumenal part of the complex is transduced to the stromal site where the catalytic domain of STT7/STN7 is located. Several hypotheses have been conceived to address this point: Vener and colleagues<sup>749</sup> proposed that the putative transmembrane helix of the kinase could directly sense PQH₂ binding to the luminal site, leading to kinase activation. Consistent with this possibility, Shapiguzov and colleagues found that the N terminus moiety of the kinase (which is supposed to be on the lumenal side) interacts with the transmembrane part of the ISP, based on yeast two hybrid<sup>751</sup>. Alternatively, a chlorophyll-based hypothesis has been proposed by Zito and colleagues. These authors analyzed of a *Cytb*<sub>6f</sub> mutant of *Chlamydomonas*, in which the small subunit PetL was fused to subunit IV to “transform” the *Cytb*<sub>6f</sub>, which contains seven helices in its core complex subunits (*Cytb*<sub>6</sub>- and sub IV), into a *Cytbc*₁-type of complex (which contains eight helices in its unique core complex subunit, *Cytb*). They found that while this mutation was not affecting electron flow, it was able to completely abolish state transitions<sup>756</sup>. They conclude therefore that the mutation was sterically hindering kinase docking to *Cytb*<sub>6f</sub>, i.e. that the kinase docking site was located in the proximity of the C terminus of subunit IV (where the subunit PetL was fused in the mutant). There, the tetrapyrrole ring of *Cytb*<sub>6f</sub> bound chlorophyll is exposed to the lipid solvent. Conversely, the PQ phytol chain goes deeply into the complex structure ending up in the lumenal pocket (in *Chlamydomonas*<sup>41</sup>) or in proximity of the F helix, which sandwiches the chlorophyll ring in the cyanobacterium *Mastigocladus laminosus*<sup>42</sup>. There, it could sense PQH₂ binding to its luminal site via a conformational change<sup>753</sup> and transduce the signal via its chlorophyll ring. The possible involvement of the chlorophyll ring in kinase activation was later supported by the finding that mutants affecting the environment of the chlorophyll ring in the complex were altering the kinetics of state transitions in *Chlamydomonas*<sup>695</sup>. Hasan and colleagues further developed this concept, proposing that specific lipids located on the surface of *Cytb*<sub>6f</sub> close to the chlorophyll could provide an adaptable surface for interaction with Stt7/STN7<sup>174</sup>. More recently, this hypothesis has been refined by pinpointing several residues in the stromal loop linking helices F and
G of *Cytb*<sub>6f</sub> subunit IV (where the chlorophyll ring is exposed) that are crucial for state transitions. This has led to the idea of an interaction between *Cytb*<sub>6f</sub> and the Stt7 kinase mediated by the stromal side of the complex above the chlorophyll ring <sup>696</sup>.

The second information concerns inhibition of state transitions in high-light treated *Chlamydomonas*
cells<sup>757</sup> and plants<sup>758,759</sup>. It was proposed that this inhibition would stem from redox control by
reduced stromal thioredoxins targeting a disulfide bond in the kinase<sup>758</sup>. Analysis of the Stt7/STN7
protein sequences revealed indeed the presence of two conserved Cys residues close to the N-terminal
end of this kinase, which are highly conserved and essential for kinase activity<sup>274,744</sup>. However,
mutagenesis of the Cys pair of the Stt7/STN7 kinase showed no change in the redox state of these
Cys during state transitions and high-light treatment. Based on these results, a different role for Cys
in kinase activation by the *Cytb*<sub>6f</sub> was proposed<sup>751</sup> in which these residues would allow the formation
of a dimer between two kinases bound to each monomer of the *Cytb*<sub>6f</sub> dimer via formation of
intermolecular disulfide bridges. Consistent with this idea, while no STN7 dimer was detectable *in
vivo*, it could be clearly detected in the *Arabidopsis* STN7-Cys mutants in which C1 or C2 was
stabilized<sup>760</sup>. Such dimer would be rather unstable and revert rapidly to an intramolecular disulfide
bridge in each monomer when the luminal site is empty, explaining why the kinase rapidly inactivates
upon oxidation of the PQ(H<sub>2</sub>) pool.

The molecular description of the Stt7 kinase<sup>753</sup> and its interaction with *Cytb*<sub>6f</sub> are unknown or
unclear. The Stt7 kinase has been cloned, expressed, and purified in a heterologous host. The kinase
is active *in vitro* in the presence of reductant and has been purified as a tetramer, determined by
analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry,
with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism
spectra have shown Stt7 to be mostly α-helical and document a physical interaction with *Cytb*<sub>6f</sub>
through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its
Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does
not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation *in vivo* could
result from direct interaction between Stt7 and *Cytb*<sub>6f</sub> or long-range reduction of Stt7 by superoxide,
known to be generated in *Cytb*<sub>6f</sub> by quinol oxidation. Although kinase activation on the *n* side of the
thylakoid membrane requires a transmembrane configuration of the kinase (Fig. 39), the only
hydrophobic domain of the kinase, near the N-terminus, contains 3-4 prolines. There is no known
precedent for such a transmembrane domain.
<figure><img src="image_44.png" /></figure>

Figure 39. Multistep model for the activation of the state transitions kinase by Cytb<sub>6f</sub> in photosynthetic eukaryotes. The model assumes that PQH<sub>2</sub> (red) interaction with the Q<sub>n</sub>/Q<sub>p</sub> site leads to conformational changes within the complex, likely mediated by the movement of the ISP-HD<sup>554</sup> (yellow). These changes would to promote the association between the cytochrome and the kinase responsible for state transitions (Stt7 in algae, STN7 in plants). Cytb<sub>6f</sub>-kinase interaction would be mediated by residues surrounding the chlorophyll ring, as indicated by mutagenesis studies<sup>756</sup>. Upon binding, the kinase would become active and phosphorylate the Cytb<sub>6f</sub>-associated subunit PetO. The active kinase would then be released from the cytochrome complex, in a step possibly involving PQH<sub>2</sub> oxidation, PQ release from the Q<sub>n</sub> site and conformational changes of the ISP, as suggested by the inhibitory effect of the PQ analogue stigmatellin on this step<sup>755</sup>. The active kinase would be in a dimeric state, thanks to the interaction between conserved cysteines present on every monomer (black squares)<sup>751</sup>. It would interact with LHCII promoting its phosphorylation, and migration from PSII to PSI. Alternatively, a ternary binding complex might be formed between LHCII, Cytb<sub>6f</sub> and the kinase. Finally, monomerization of the kinase would inactivate it, closing the activation cycle. See also Singh et al.<sup>753</sup>, for a further discussion.

# 11 Comments on the nomenclature used for Cytbc<sub>1</sub> and Cytb<sub>6f</sub>

In the literature there are number of differences in naming of similar structural and/or
functional elements of Cytbc<sub>1</sub> and Cytb<sub>6f</sub>. This section contains short comments on nomenclature to
clarify the potentially confusing terms and, when needed, explains how they are used in this review.

**Complexes:** The term „Cyt-bc" refers to both Cytbc<sub>1</sub> and Cytb<sub>6f</sub> as enzymes belonging to one group traditionally named cytochromes bc. This is how it is used in the review. Based on phylogenic analysis, it has been proposed to rename this group as "Rieske/b complexes", as it would better reflect the key and evolutionary unchanged subunits.<sup>761</sup> However this name, in spite of being scientifically justified, did not gain popularity in the literature.

In mitochondrial electron transport chain, Cytbc₁ is also named „mitochondrial complex III” or in short ”complex III”.
**Quinone binding catalytic sites:** The quinol oxidation site is named the "Q<sub>o</sub> site" (in the case of Cytbc<sub>1</sub>, in particular bacterial Cytbc<sub>1</sub>) or "Q<sub>p</sub> site" (in the case of Cytb<sub>6f</sub> and sometimes eukariotic Cytbc<sub>1</sub>/complex III). The same rule applies for the quinone reduction site which is named either the "Q<sub>i</sub> site" (Cytbc<sub>1</sub>) or the "Q<sub>n</sub> site" (Cytb<sub>6f</sub> and Cytbc<sub>1</sub>/complex III). The subscripts "o" and "i" were originally used by Mitchell to indicate "out" and "in", respectively, as the direction of proton transfers catalyzed by the respective sites. The subscripts "p" and "n" refer to "positive" and "negative" side of the membrane. In brief: Q<sub>o</sub> = Q<sub>p</sub> while Q<sub>i</sub> = Q<sub>n</sub>.

In this review we use $"Q_o/Q_p"$ and $"Q_i/Q_n"$ when referring to the sites in both Cytbc<sub>1</sub> and Cytb<sub>6f</sub>; $"Q_o"$ and $"Q_i"$ when referring specifically to Cytbc<sub>1</sub>; $"Q_p"$ and $"Q_n"$ when referring specifically to Cytb<sub>6f</sub>.

**Cofactor chains:** In Cytbc<sub>1</sub> the "high-potential chain" is alternatively named the "c-chain" and the "low-potential chain" is the "b-chain".

**Protein subunits:** In Cytb<sub>6f</sub>, the assembly of two subunits: Cytb<sub>6</sub> and Subunit IV corresponds to one larger subunit – Cytb of Cytbc<sub>1</sub>.

**Cofactors:** The *b* hemes in Cytbc<sub>1</sub> and Cytb<sub>6f</sub> are often named differently. Hemes *b* in Cytbc<sub>1</sub> are usually named *b*<sub>H</sub> and *b*<sub>L</sub> (to denote higher and lower *E*<sub>m</sub> of the heme, respectively). Their counterparts in Cytb<sub>6f</sub> are named *b*<sub>n</sub> and *b*<sub>p</sub> (to denote proximity of the heme to negative and positive side of the membrane, respectively). In brief: *b*<sub>H</sub> = *b*<sub>n</sub> while *b*<sub>L</sub> = *b*<sub>p</sub>.

In this review we use heme *b*<sub>H</sub>/*b*<sub>n</sub> and *b*<sub>L</sub>/*b*<sub>p</sub> when referring to hemes in Cytbc<sub>1</sub> and Cytb<sub>6f</sub>. Hemes *b*<sub>H</sub> and *b*<sub>L</sub> are used when referring specifically to hemes of Cytbc<sub>1</sub>. Hemes *b*<sub>n</sub> and *b*<sub>p</sub> are used when referring specifically to hemes of Cytb<sub>6f</sub>.

The atypical *c*-type heme present in Cytb<sub>6f</sub> is designated using different symbols such as *c*<sub>n</sub> and *c*<sub>i</sub> and also *c*<sub>x</sub>. All these symbols describe the same heme.

**Other:** When describing the motion of ISP-HD, the "Q<sub>o</sub>-position" or "b-position" of ISP-HD refers to the same position of ISP-HD (when it is bound at the Q<sub>o</sub> site/cytochrome *b* or the Q<sub>p</sub> site/cytochrome *b*<sub>6</sub>).

# 12 References
(1) Berry, E. A.; Guergova-Kuras, M.; Huang, L.; Crofts, A. R. Structure and Function of Cytochrome Bc Complexes. *Annu. Rev. Biochem.* **2000**, *69*, 1005–1075.

(2) Crofts, A. R. The Cytochrome *Bc*₁ Complex: Function in the Context of Structure. *Annu. Rev. Physiol.* **2004**, *66*, 689–733.

(3) Borek, A.; Ekiert, R.; Osyczka, A. Advances in Understanding Mechanism and Physiology of Cytochromes *Bc*. In *Mechanisms of primary energy transduction in biology*; Wikström, M., Ed.; Royal Society of Chemistry: London, **2018**; pp 192–214.

(4) Cramer, W. A. Structure–Function of the Cytochrome *b*6f Lipoprotein Complex: A Scientific Odyssey and Personal Perspective. *Photosynth. Res.* **2019**, *139*, 53–65.

(5) Kirchhoff, H.; Li, M.; Puthiyaveetil, S. Sublocalization of Cytochrome *b*6f Complexes in Photosynthetic Membranes. *Trends Plant Sci.* **2017**, *22*, 574–582.

(6) Schöttler, M. A.; Tóth, S. Z.; Boulouis, A.; Kahlau, S. Photosynthetic Complex Stoichiometry Dynamics in Higher Plants: Biogenesis, Function, and Turnover of ATP Synthase and the Cytochrome B6f Complex. *J. Exp. Bot.* **2015**, *66*, 2373–2400.

(7) Tikhonov, A. N. The Cytochrome B6f Complex at the Crossroad of Photosynthetic Electron Transport Pathways. *Plant Physiol. Biochem.* **2014**, *81*, 163–183.

(8) Tikkanen, M.; Grieco, M.; Nurmi, M.; Rantala, M.; Suorsa, M.; Aro, E. M. Regulation of the Photosynthetic Apparatus under Fluctuating Growth Light. *Philos. Trans. R. Soc. B Biol. Sci.* **2012**, *367*, 3486–3493.

(9) Cruz-Gallardo, I.; Díaz-Moreno, I.; Díaz-Quintana, A.; De La Rosa, M. A. The Cytochrome F-Plastocyanin Complex as a Model to Study Transient Interactions between Redox Proteins. *FEBS Lett.* **2012**, *586*, 646–652.

(10) Nelson, N. Photosystems and Global Effects of Oxygenic Photosynthesis. *Biochim. Biophys. Acta - Bioenerg.* **2011**, *1807*, 856–863.

(11) Nowaczyk, M. M.; Sander, J.; Grasse, N.; Cormann, K. U.; Rexroth, D.; Bernát, G.; Rögner, M. Dynamics of the Cyanobacterial Photosynthetic Network: Communication and Modification of Membrane Protein Complexes. *Eur. J. Cell Biol.* **2010**, *89*, 974–982.

(12) Trebst, A. Inhibitors in the Functional Dissection of the Photosynthetic Electron Transport System. *Photosynth. Res.* **2007**, *92*, 217–224.

(13) Sarewicz, M.; Osyczka, A. Electronic Connection between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling. *Physiol. Rev.* **2015**, *95*, 219–243.

(14) Allen, J. F. Cytochrome B6f: Structure for Signalling and Vectorial Metabolism. *Trends Plant Sci.* **2004**, *9*, 130–137.

(15) Choquet, Y.; Wostrikoff, K.; Girard-Bascou, J.; Wollman, F.-A. Assembly-Controlled Regulation of Chloroplast Gene Translation. *Biochem. Soc. Trans.* **2001**, *29*, A51–A51.