Source: https://pubs.rsc.org/en/content/articlehtml/2019/mt/c8mt00218e
Timestamp: 2019-04-24 07:54:24+00:00

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In bacteria, copper (Cu) is often recognised for its potential toxicity and its antibacterial activity is now considered a key component of the mammalian innate immune system. Cu ions bound in weak sites can catalyse harmful redox reactions while Cu ions in strong but adventitious sites can disrupt protein or enzyme function. For these reasons, the outward transport of Cu from bacteria has received significant attention. Yet, Cu is also a bacterial nutrient, required as a cofactor by enzymes that catalyse electron transfer processes, for instance in aerobic and anaerobic respiration. To date, the inward flow of this metal ion as a nutrient and its insertion into target cuproenzymes remain poorly defined. Here we revisit the available evidence related to bacterial nutrient Cu trafficking and identify gaps in knowledge. Particularly intriguing is the evidence that bacterial cuproenzymes do not always require auxiliary metallochaperones to insert nutrient Cu into their active sites. This review outlines our effort to consolidate the available experimental data using an established energy-driven model for metalation.
Fig. 1 General energy-driven model for the insertion of Cu into cuproenzymes. The relative energy for each Cu-binding site, whether in the buffer (B1, B2, B3), cuproprotein (P), or metallochaperone (M) is shown. Curved arrows represent the forward transfer of Cu from one binding site to another while double-headed arrows represent the energy barrier that must be overcome. Several scenarios are depicted: (a) upon entry into cells, Cu fills the buffer by stepwise transfer from high energy or low affinity sites (denoted as B3) to low energy or high affinity sites (denoted as B1) in the buffer through stochastic exchange reactions. (b) Direct transfer of Cu from the buffer (in this example the mid-affinity or mid-energy site B2) to a cuproprotein (P). (c) Transfer of Cu from the mid-affinity buffer (B2) to a cuproprotein (P) via a metallochaperone (M). Equations representing these equilibria are shown on the right. (d) During conditions of Cu starvation, low affinity or high energy sites in the buffer (B3) start to empty, leaving only Cu that is bound in high affinity or low energy buffer sites (B1). Onward transfer of Cu from this low energy buffer to the cuproprotein (P) is shown with a high energy barrier. (e) During conditions of Cu stress, the excess Cu starts to fill the weaker sites in the buffer start (B3). Onward transfer from this high energy buffer to the cuproprotein (P) is shown, requiring a lower activation energy.
Cu sits at the top of the Irving–Williams series and hence metalation of cuproenzymes is normally an endergonic or thermodynamically favourable process. By the same principle, Cu can also partition into stable sites in the wrong protein, leading to enzyme inactivation and bacterial poisoning. To minimise mis-metalation, cells employ metallochaperones that are thought to shuttle (or “chaperone”) the Cu ion from import pumps to target cuproproteins. Such pathways are well described for the eukaryotic cytosol and organelles.5–9 For prokaryotes, discussions of Cu homeostasis have revolved mainly around Cu tolerance,10–12i.e. removal of excess Cu from the cell under conditions of Cu surplus, when the cellular Cu buffer is “full”. By contrast, trafficking of nutrient copper, particularly when the buffer is “empty”, is less understood.
Cuproenzymes are thought to have evolved after the appearance of atmospheric O213 and so they are typically involved in reactions with oxygen and oxygen-containing species. In prokaryotes, Cu is a major nutrient for aerobic respiration (via haem-Cu oxidases in the electron transport chain), anaerobic respiration (via nitrous oxide reductases and Cu-containing nitrite reductases in the denitrification pathway), and removal of toxic reactive oxygen species (via Cu,Zn-superoxide dismutase). Intriguingly, the Cu-dependent enzymes in the aforementioned pathways are all localised to the bacterial envelope (i.e. in the periplasm of Gram-negative bacteria or on the surface of Gram-positive bacteria). Indeed, with the exception of plastocyanin and cytochrome oxidase in cyanobacteria, cuproproteins are not known to exist inside the bacterial cytoplasm. This apparent compartmentalisation of Cu to the extracytoplasmic space may represent a mechanism for balancing the physiological advantages of using nutrient Cu in catalysis while protecting against its potential toxicity. Indeed, Cu is generally considered to be more toxic in the cytoplasm and thus must be buffered at a lower availability (i.e. bound by higher affinity or lower energy sites in the buffer) relative to the extracytoplasmic space.
Metals in the bacterial envelope are readily exchangeable with the extracellular environment, for example via passive diffusion across outer membrane porins in Gram-negative organisms.14 Hence, fine control of metalation in this compartment may be more challenging than in the cytoplasm. This is considered particularly problematic for metalloproteins that are translocated via the Sec general secretory pathway and thus are folded (and metalated) in the extracytoplasmic space.15 By contrast, metalloproteins that are Tat substrates fold inside the cytoplasm and, at least in some cases, obtain their cognate metal prior to secretion.15 In the case of Cu, recent examination of the periplasmic multicopper oxidase CueO from Escherichia coli demonstrated that removal of the Tat signal sequence and expression of CueO in the cytoplasm led to isolation of only the apo-enzyme.16 In fact, all bacterial cuproproteins for which the steps of Cu insertion have been identified (detailed in this review) are thought to become metalated outside the cytoplasm, regardless of the translocation mechanism of the protein scaffold. One explanation is that the Cu affinities of these cuproenzymes are compatible with the buffered availability of Cu in the bacterial envelope but incompatible with that of the cytoplasm. In addition, the oxidation state of Cu in the buffer and the oxidation state preferred by the enzyme might further define extracytoplasmic metalation of cuproproteins.
What is the source of nutrient Cu for cuproenzymes in the bacterial envelope? In the simplest model, a buffered pool of Cu in the extracytoplasmic space acts as the Cu supplier. The molecular nature of this Cu buffer is presently unknown. It has been long assumed that thiols like glutathione (GSH) buffer Cu in the cytoplasm.17 There is also evidence that GSH is exported to the periplasm of Gram-negative organisms,18 and so it can presumably also buffer Cu in this compartment. However, the affinity of GSH for Cu is orders of magnitudes weaker when compared to those of bacterial Cu sensors in the cytoplasm19,20 or nutrient Cu metallochaperones in the periplasm.21 These relative values suggest that, despite its high intracellular concentrations, GSH would constitute a high energy buffer, filled only when an excess of Cu is available. By contrast, the identity of the low energy or high affinity buffer that contributes to normal Cu nutrition is unknown. Nevertheless, metalloproteomics examination of periplasmic extracts from Salmonella enterica sv. Typhimurium22 and Synechocystis23 indicated that periplasmic Cu is largely bound either to Cu metallochaperones or to unidentified low molecular weight proteins.
Regardless of the precise identity of the extracytoplasmic Cu buffer, it is presumably filled by Cu from the extracellular environment (Fig. 2). This exchange of Cu may occur via passive diffusion through porins14,24 or other unidentified mechanisms.25 Active uptake of Cu is also known to occur, for example via TonB-dependent receptors26 or via classical siderophores27 and Cu-binding metallophores (“chalkophores”) such as yersiniabactin and methanobactin.28–30 Once the buffer is filled by Cu, provided that the affinities of the cuproenzymes are higher than the affinity of the buffer, Cu will flow down the thermodynamic gradient and ultimately insert into target enzymes (Fig. 1b). Yet, there is now mounting evidence that Cu-exporting P-type ATPases embedded in the cytoplasmic membrane are involved in metalating extracytoplasmic cuproproteins.23,31,32 The implication is that nutrient Cu ions are trafficked through the cytoplasm en route to the extracytoplasmic targets and, if so, this must be a vital process for Cu homeostasis. When combined with the dearth of known cytoplasmic Cu importers, this apparently circuitous routing of Cu is one of the most puzzling aspects of nutrient Cu handling in bacteria.
Fig. 2 General model for the insertion of Cu into NosZ. [Cu-Be], [Cu-Bc], and [Cu-Bp] are buffered pools of Cu in the extracellular space, cytoplasm, and periplasm, respectively. In this model, [Cu-Bp] is filled by [Cu-Be] either via the TonB-dependent receptor NosA or possibly via direct exchange across outer membrane porins in NosA-deficient organisms. [Cu-Bp] is also filled by [Cu-Bc] via the P-type ATPase CtpA. How [Cu-Bc] is generated is unknown. The CuZ site in NosZ acquires Cu from [Cu-Bp] either directly or via the metallochaperone NosL, and this process is likely coupled with insertion of sulfur (S) by NosDFY. How the CuA site obtains Cu is unknown but this process likely resembles mechanisms for CuA assembly in haem-Cu oxidases. IM, inner membrane; OM, outer membrane.
Our research groups have studied bacterial Cu tolerance for several years and have recently begun to investigate nutrient Cu handling, specifically in pathogenic Neisseria. This prompted us to revisit existing literature related to bacterial nutrient Cu trafficking and identify gaps in knowledge. We were particularly intrigued by the evidence that bacterial cuproenzymes do not always require auxiliary metallochaperones to insert nutrient Cu into their active sites. This review outlines our effort to consolidate the available experimental data by expanding an established energy-driven model for Cu trafficking.2 We focus on four major families of bacterial cuproenzymes: (1) nitrous reductases, (2) nitrite reductases, (3) Cu,Zn-superoxide dismutases, and (4) haem-Cu oxidases, and pay particular attention to the precise steps of Cu insertion. The genomic context and genetic distribution, structural features and properties of the Cu centres in the enzymes (and in the associated metallochaperones), as well as kinetic properties of these enzymes are already subjects of numerous excellent reviews and so will not be covered in detail.
Consistent with its high demand for Cu, NosZ activity in denitrifying organisms is greatly influenced by extracellular Cu levels.38–40 During conditions of Cu deficiency, NosZ activity decreases and N2O accumulates. This Cu-dependent regulation of NosZ occurs at the post-translational level, i.e. by modulating occupancy of the Cu centres. Growth in Cu-deficient conditions leads to production of NosZ in an inactive form. However, N2O reductase activity is restored by addition of exogenous Cu without the need for new protein synthesis.39 In bacteria possessing the typical NosZ, increases in extracellular Cu levels also induce the expression of nosZ. This requires at least one factor, the flavoprotein NosR, although the molecular details are yet to be elucidated.40,41 The nosR gene is not found in genomes encoding atypical NosZ,33 and whether Cu regulates nosZ transcription in these organisms is unknown.
The current models for CuZ and CuA biogenesis suggest that these Cu centres are assembled in the periplasm following secretion of the protein, for both the typical and atypical NosZ, regardless of the translocation mechanism (Fig. 2). Homologous expression of NosZ in the cytoplasm results in the production of neither the CuZ nor the CuA centre.42 Assembly of CuZin vivo requires NosDFY, an ABC-type transporter that may transport sulfur (Fig. 2)43 although this is yet to be confirmed experimentally. This requirement for NosDFY appears to be obligate and the genetic clustering of nosZ with nosDFY is absolutely conserved in all sequenced genomes that are currently available.33 N2O respiration is abolished if any of the nosDFY genes is mutated and this defect is not restored by addition of extracellular Cu.44,45 In addition, NosZ isolated from nosDFY-deficient strains contains only the CuA centre,46–48 indicating that NosDFY may not be required to assist CuA assembly.
Insertion of nutrient Cu into the CuZ cluster in vivo is likely facilitated by NosL, a small lipoprotein that is anchored to the outer membrane (Fig. 2). The soluble periplasmic domain of NosL binds one Cu(I) ion in vitro but its affinity has not been determined.49 The Cu ligands include one Cys and one Met, presumably from a conserved Cys-X-Met motif near the N-terminus.50,51 The third ligand, likely from a His residue, is yet to be identified, and no obvious candidate is found from analysis of amino acid sequences. Whether NosL delivers Cu(I) to NosDFY or directly to NosZ, whether metalation is coupled to sulfur insertion, and whether NosL assists in assembly of the CuA centre are yet to be established. None of the nos cluster genes appears to be essential for CuA assembly. Nevertheless, denitrifying organisms often possess additional Cu metallochaperones like Sco and PCuAC (described below), which may metalate the CuA sites in NosZ, but this remains to be elucidated.
How does CuZ obtain nutrient Cu in the absence of NosL? There is a proposal that other Cu metallochaperones such as PCuAC (described below) can compensate, although this is yet to be tested experimentally. An alternative, and arguably simpler, hypothesis is that the CuZ site acquires Cu directly from the extracytoplasmic Cu buffer (Fig. 2). This reaction is thermodynamically favourable (“downhill” or exergonic) as long as the affinity of the CuZ scaffold for Cu is higher than the affinity of the buffer (i.e. the bound Cu ion in CuZ is lower in energy or more stable than is Cu in the extracytoplasmic buffer) (Fig. 1b). NosL may provide an “intermediate buffer” (with intermediate Cu affinities) that lowers the overall energy barrier for the transfer of Cu from the extracytoplasmic buffer to the CuZ scaffold, with Cu-NosL acting as a reaction intermediate (Fig. 1c). In this scenario, the absence of NosL would not affect the Cu occupancy of NosZ, provided that the buffered Cu availability is sufficiently high (i.e. Cu is bound by high energy or low affinity sites in the buffer) and the barrier for Cu transfer to NosZ is sufficiently low. NosL would become more important in Cu-deficient conditions, when the buffered Cu availability decreases (i.e. Cu is bound by low energy or high affinity sites in the buffer) and thus, presumably, the barrier for onward Cu transfer to NosZ increases (Fig. 1d).
Regardless of the precise role for NosL, the question remains: what is the source of the buffered Cu in the extracytoplasmic space? In the simplest model, this buffer is filled directly by Cu from the extracellular environment (Fig. 2). In some, but not all, denitrifying Gram-negative organisms, N2O respiration during conditions of Cu limitation requires NosA, a TonB-dependent receptor that may increase uptake of Cu into the periplasm (Fig. 2).26,46,54,55 Intriguingly, there is also evidence that the extracytoplasmic pool of Cu is filled by supply from the cytoplasm. NosZ activity in vivo was shown to depend on CtpA, a P-type ATPase that resembles known bacterial Cu-efflux transporters (Fig. 2).31 Mutation of ctpA leads to decreased NosZ activity but enzyme activity is restored by addition of Cu to the extracellular medium. This exogenous Cu presumably fills the extracytoplasmic Cu buffer, which in turn metalates NosZ (Fig. 2). If direct metalation of NosZ by the extracytoplasmic Cu buffer is possible in the ΔctpA mutant, why nutrient Cu must first be routed through the cytoplasm in the wild type organism appears a major conundrum.
T1 and T2 Cu centres are readily reconstituted by Cu salts in vitro and so insertion of Cu into NirK in vivo was previously assumed to require no accessory metallochaperones. However, a recent genetic screen identified that a soluble periplasmic Cu-binding protein, AccA, is required for metalating NirK (AniA) in pathogenic Neisseria (Fig. 3).60 AccA is a homologue of PCuAC, a metallochaperone that is thought to aid assembly of CuA and CuB centres in haem-Cu oxidases61–64 (described below). Like PCuAC, AccA binds one Cu(I) ion with a high apparent affinity,60 although precise quantification is still awaited. Conserved Met and His residues are likely involved in binding Cu(I). AccA also binds one additional Cu ion in the Cu(II) oxidation state.60 Several candidate ligands for Cu(II) are present in the His- and Met-rich C-terminus but their identities are yet to be determined. Programmes in our research groups are currently ongoing to determine which of the two bound Cu ions in AccA is loaded to which of the two Cu sites in AniA.
Fig. 3 General model for the insertion of Cu into AniA (NirK). [Cu-Be], [Cu-Bc], and [Cu-Bp] are buffered pools of Cu in the extracellular space, cytoplasm, and periplasm, respectively. The T1 and T2 sites in AniA acquire Cu from [Cu-Bp] either directly or via the metallochaperone AccA (PCuAC). [Cu-Bp] is likely filled by [Cu-Be] via direct exchange across outer membrane porins. Whether an outer membrane importer or a cytoplasmic exporter is involved in filling [Cu-Bp] is yet to be determined. IM, inner membrane; OM, outer membrane.
Mutants lacking accA generate wild type amounts of AniA but fail to reduce NO2−, suggesting that AniA is produced in the apo- or incorrectly metalated form. Consistent with this view, reduction of NO2− resumes, albeit only partially, upon addition of Cu salts into the extracellular media.60 Assuming that no other unidentified Cu trafficking pathway compensates for AccA, the observed recovery of AniA activity by exogenous Cu is consistent with the proposal that that this enzyme is metalated directly by a buffered Cu pool in the periplasm (Fig. 3). This reaction is energetically downhill as long as the affinity of AniA for Cu is higher than the affinity of the buffer (i.e. the bound Cu in AniA is more stable or less energetic) (Fig. 1b). As hypothesised earlier for NosL, the function of AccA may be to act as an intermediate buffer that lowers the overall energy barrier for Cu exchange and thus functionally “catalyses” the transfer of Cu from the buffer to the T1 and/or T2 sites of AniA (Fig. 1c). In the absence of AccA, provided that the buffered Cu availability is sufficiently high (i.e. Cu is bound by high energy or low affinity sites in the buffer), the barrier for onward Cu transfer decreases, and AniA becomes metalated (Fig. 1e).
Like pathogenic Neisseria, some NirK-containing organisms also possess the metallochaperone Sco.66,67 Together with PCuAC, Sco is thought to facilitate assembly of CuA and CuB centres in haem-Cu respiratory oxidases (described below). Whether Sco is required for inserting Cu into T1 and T2 sites of NirK is not known. Likewise, whether a Cu importer such as NosA from P. stutzeri or a Cu-exporting P-type ATPase such as CopA in pathogenic Neisseria is involved in metalating AniA or NirK is yet to be examined (Fig. 3).
Fig. 4 General model for the insertion of Cu into SodC. [Cu-Be], [Cu-Bc], and [Cu-Bp] are buffered pools of Cu in the extracellular space, cytoplasm, and periplasm, respectively. [Cu-Bp] is likely filled by [Cu-Be] via direct exchange across outer membrane porins. Whether an outer membrane importer is involved in this process is yet to be established. [Cu-Bp] is also filled by [Cu-Bc] via the P-type ATPases CopA or GolT. How [Cu-Bc] is generated is unknown. The T2 Cu site in SodC acquires Cu from [Cu-Bp] either directly or via the metallochaperone CueP. IM, inner membrane; OM, outer membrane.
Fig. 5 A general energy-driven model for the insertion of Cu into the wrong proteins (mismetalation). The relative energy for each Cu-binding site, whether in the buffer (B2, B3), Cu-binding metallochaperone (M), or a non-native adventitious protein (X) is shown. Curved arrows represent the forward transfer of Cu from one binding site to another. Several scenarios are depicted: (a) protein X, which is not a Cu-binding protein, binds Cu with an affinity that is weaker than that of the B2 buffer (i.e. the Cu-X complex is less stable or is more energetic than is the Cu-B2 complex). Hence, during normal Cu conditions, Cu transfer from buffer B2 to protein X is thermodynamically unfavourable (straight upward arrows), and X is not mismetalated by Cu. (b) During conditions of Cu stress, excess Cu enters cells and begins to fill low affinity or high energy sites in the buffer (B3). If this site is sufficiently high in energy, Cu will transfer out of the buffer into protein X, causing mismetalation. This transfer of Cu is now thermodynamically downhill and favourable (straight downward arrows). (c) Expression of a Cu-binding metallochaperone (M) during Cu stress conditions provides alternative, high-affinity or low energy but, more importantly, specific sites for Cu. Cu is thus transferred out of protein X and mismetalation is alleviated.
Another mechanism to maintain SodC in its metalated form may involve control of the oxidation state of Cu. The ΔcueP mutant is Cu-sensitive only during anaerobic growth conditions.77 In the presence of O2, Cu(I) is removed from the buffer via oxidation to Cu(II) by the cuprous oxidase CueO,84,85 and thus additional buffering of Cu(I) by CueP may not be necessary. In addition, Cu in the resting form of SodC exists in the Cu(II) state. In vitro, this bound Cu(II) ion does not re-partition into apo-CueP.32 Thus, overexpression of CueP in vivo is unlikely to lead to de-metalation of SodC, at least under aerobic growth conditions, when SodC activity is essential.86 During anaerobic growth, when SodC is not required, extracytoplasmic reductants may reduce the Cu(II) ion in SodC and subsequent back-transfer of Cu(I) to CueP is plausible, although not yet demonstrated.
Metalation of SodC in vivo also depends, at least partly, on outward transport of Cu from the cytoplasm to the periplasm via either one of the two, functionally redundant, Cu efflux pumps in S. Typhimurium, CopA and GolT (Fig. 4). SodC isolated from mutant bacteria lacking both P-type ATPases contains only the Zn centre but readily acquires Cu upon addition of Cu(II) salts into cell-free extracts.32 This finding may further highlight the importance of the correct oxidation state for Cu. CopA and GolT transport Cu in the reduced Cu(I) form, and the relative affinities of the periplasmic domains of the P-type ATPases, the periplasmic buffer, CueP, and SodC for Cu(I) may be ordered such that metalation of SodC with Cu(I) is thermodynamically favourable. However, as already mentioned earlier, the T2 site in SodC is also competent to acquire Cu(II), at least in vitro.32In vivo, one possibility is that the buffered Cu(II) availabilities (or energies) in the periplasm are low and hence insertion of Cu(II) into SodC may be a thermodynamically uphill or unfavourable process. Measurements of the affinities of CueP and SodC each for Cu(I) and Cu(II), and comparisons with the buffered availabilities of Cu(I) and Cu(II) in the periplasm would be informative.
Of interest in this review are the precise steps of Cu insertion into CuB and CuA. These processes are most studied for mitochondrial cytochrome c oxidase (COX) in eukaryotes.88,89 Given the endosymbiotic bacterial origin of mitochondria, the mechanisms for metalation of mitochondrial COX and bacterial haem-Cu oxidases likely share some universal features. The bacterial metallochaperones involved in CuB and CuA assembly, namely Sco,64,90–94 PCuAC,61–63,65,95 or Cox11p,96–100 are, again, localised to the extracytoplasmic space (Fig. 6). These metallochaperones are structurally and functionally analogous to their eukaryotic counterparts (PCuAC acts as a functional Cox17 homologue). However, unlike the eukaryotic system, the precise contribution of each protein in the assembly of bacterial CuAvs. CuB centres and the sequence of Cu insertion events remain poorly defined and, bafflingly, appear to be organism-dependent.
Fig. 6 General model for the insertion of Cu into the CuB site into cytochrome cbb3 oxidase. Only the active site subunits CcoNOP are shown. This model may broadly apply to insertion of Cu into the CuA site in other haem-Cu oxidases. [Cu-Be], [Cu-Bc], and [Cu-Bp] are buffered pools of Cu in the extracellular space, cytoplasm, and periplasm, respectively. The CuB site in CcoN (and/or CuA site in other haem-Cu oxidases) may obtain nutrient Cu directly [Cu-Bp] or via the periplasmic Cu metallochaperones PCuAC and Sco. Based on studies on CuA assembly, Sco may also act as a thiol–disulfide reductase that maintains either the Cu ion or the CuB (or CuA) Cys ligands in their reduced forms. Upstream reductases such as TlpA may provide the reducing equivalents. Supply of Cu to [Cu-Bp] could occur by direct exchange across outer membrane porins or via an as yet unidentified importer. The MFS transporter CcoA supplies Cu to [Cu-Bc], with reduction from Cu2+ to Cu+ occurring either during transit or spontaneously in the reducing environment of the cytoplasm. Cu is routed back to the periplasm to fill [Cu-Bp] via the P-type ATPase CcoI. IM, inner membrane; OM, outer membrane.
As mentioned earlier, not all bacterial haem-Cu oxidases contain a CuA centre. However, in contrast to the relative wealth of information available for CuA, current understanding of CuB assembly remains limited, mainly because the location of CuB deep within a transmembrane domain has largely precluded in vitro studies. Nevertheless, as discussed below, there is mounting in vivo evidence that Sco and PCuAC are also involved in CuB assembly, at least in some organisms. The mechanism may parallel that for CuA although the precise details still need investigation. In addition, the bacterial homologue of mitochondrial Cox11, Cox11p, has been implicated in forming bacterial CuB centres in vivo.98,100 Whether bacterial Cox11p coordinates its function with PCuAC and/or Sco is unknown.
Like the other extracytoplasmic cuproenzymes described in this review, haem-Cu oxidases also appear to utilise nutrient Cu that has been routed via the cytoplasm, first via a major facilitator superfamily (MFS)-type transporter named CcoA that putatively imports Cu into the cytoplasm112,113 and subsequently via a Cu efflux pump (CcoI or CtpA)31,111,114,115 (Fig. 6). Deletion of each of these transporters leads to decreases in the activities of CuB and/or CuA-containing cytochrome oxidase activities, but these are, to some extent, alleviated by supplementation with Cu salts. This apparent routing of Cu through the intracytoplasmic compartment to metalate an extracytoplasmic cuproenzyme is one of the least understood aspects of nutrient Cu trafficking but, if it does occur, must represent a vital process in bacterial Cu homeostasis.
Among the six, first-row d-block transition metal ions that are considered as bacterial nutrients (Mn, Fe, Co, Ni, Cu, Zn), Cu is often highlighted for its potential toxicity. Cu ions bound in weak or high energy or unstable sites can catalyse harmful redox reactions, while Cu ions in strong, low energy or stable but non-native (adventitious) sites (mismetalation) can disrupt protein or enzyme function. While the outward transport of Cu as a bacterial poison has received significant attention from the metallomics community, inward flow of this metal ion as a bacterial nutrient remains less defined. Confounding this issue, known bacterial Cu importers and Cu-binding metallophores are still exceedingly rare and, as described in this review, while they are relatively more common, nutrient Cu metallochaperones are often functionally redundant.
The apparent redundancy of Cu metallochaperones may be rationalised by the energy-driven model, in which target cuproenzymes obtain nutrient Cu directly from a buffered Cu pool via “downhill” or exergonic associative exchange reactions (Fig. 1b). It is our view that this model can universally rationalise all the available experimental evidence for the metalation of cuproenzymes in different bacterial organisms. In this model, the apo-metallochaperones can be considered as intermediate buffers or functional catalysts that lower the energy barrier for Cu transfer and regulate the flow of Cu down the thermodynamic gradient (Fig. 1c). The Cu-bound form of the metallochaperone thus represents a thermodynamic local minimum that limits “sideway” flows of Cu into adventitious sites (Fig. 1c). Hence, these metallochaperones are not obligate components for Cu homeostasis but are nonetheless able to provide alternative and more efficient routes for metalation during Cu nutrition, particularly when extracellular Cu is limiting, and for preventing (or correcting) mismetalation during Cu poisoning. This “intermediate buffering” function for Cu metallochaperones has indeed been proposed previously81–83 but how these metallochaperones lower the energy barrier for Cu transfer remains to be determined.
A key advantage of this model is that, in organisms where the metallochaperone is absent, there is no need to describe elaborate backup or compensatory mechanisms. Instead, the main considerations would be the oxidation state of Cu, as well as the relative amounts and Cu affinities of the target cuproenzymes, of the metallochaperones, and of the extracytoplasmic buffer. Differences in these properties may explain why periplasmic cuproproteins do not acquire Cu when expressed homologously in the cytoplasm. If the affinities of the cytoplasmic buffer for Cu are higher than the affinities of the cuproenzymes (i.e. bound Cu in the buffer is less energetic or more stable), transfer of Cu out of the buffer would be thermodynamically uphill or endergonic. Hence, knowledge of the relative tunings of extracytoplasmic buffer components compared to the cytoplasm becomes equally important.
We thank Peter Chivers and Nigel Robinson (Department of Biosciences, Durham University) for enlightening discussions related to metal buffers. Louisa Stewart is supported by a Research Excellence Academy Studentship funded by Newcastle University. Denis Thaqi is supported by a Research Training Program Scholarship (Department of Education and Training, Commonwealth of Australia). Bostjan Kobe is supported by the National Health and Medical Research Council (NHMRC) Program Grant 1071659 and NHMRC Principal Research Fellowship (APP1110971). Kevin Waldron is supported by a Sir Henry Dale Fellowship (098375/Z/12/Z) co-funded by the Wellcome Trust and the Royal Society. Karrera Djoko is supported by the Royal Society Research Grant (RSG\R1\180044).
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† These authors contributed equally to this work and the names are listed alphabetically.

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