# A scaffold for quinone channeling between membrane and soluble bacterial oxidoreductases

M. Broc, M. Cherrier, A. Uzel, R. Arias-Cartin, P. Arnoux, G. Brasseur, F. Seduk, B. Guigliarelli, P. Legrand, F. Pierrel, et al.

► To cite this version:

M. Broc, M. Cherrier, A. Uzel, R. Arias-Cartin, P. Arnoux, et al. A scaffold for quinone channeling between membrane and soluble bacterial oxidoreductases. Nature Structural and Molecular Biology, 2025, 32, pp.2196-2202. 10.1038/s41594-025-01607-4. hal-05229188

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# A scaffold for quinone channeling between membrane and soluble bacterial oxidoreductases

Received: 22 March 2024

Accepted: 27 May 2025

Published online: 25 August 2025

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M. Broc<fnref n="1" />,¹⁰, M. V. Cherrier©²,¹⁰, A. Uzel³, R. Arias-Cartin<fnref n="1" />,⁹, P. Arnoux©⁴, G. Brasseur<fnref n="1" />, F. Seduk<fnref n="1" />, B. Guigliarelli©³, P. Legrand©⁵, F. Pierrel©⁶, G. Schoehn©⁷, M. J. Maté⁸, L. Martin©², S. Grimaldi³, Y. Nicolet©²✉, A. Magalon©<fnref n="1" />✉ & A. Walburger©<fnref n="1" />✉

Redox processes are at the heart of energetic metabolism that drives life on earth. By extension, complex and efficient electron transfer wires are necessary to connect the various metabolic pathways that are often located in distinct cellular compartments. Here, we uncovered a structural module that enables channeling of quinones from the membrane to various water-soluble redox catalytic units in prokaryotes. Using X-ray crystallography and cryo-electron microscopy, we determined the structure of the unusual bacterial formate dehydrogenase ForCE that contains four ForC catalytic subunits docked around a membrane-associated tetrameric ForE central scaffold. In the latter, a conserved domain that we propose to name helical membrane plugin (HMP) was identified as essential to link formate oxidation, in *Bacillus subtilis*, to the aerobic respiratory chain. Our bioinformatic analysis indicates that this HMP is associated with different quinone-reducing oxidoreductases, highlighting its broad importance as a functional unit to wire electrons between a given catalytic redox center and the quinone pool.

Biological redox processes are fundamental to life as they afford various bioenergetic pathways such as respiration and carbon and nitrogen fixation<fnref n="1" />. These reactions have a crucial role in major geochemical cycles by connecting cells to key atmospheric gasses such as N₂, CO₂ or H₂. The enzymes catalyzing these redox reactions primarily use cofactors containing transition metals and are often considered as ancient enzymes participating in the early reactions that contributed to the origin of life²⁻⁵. Interestingly, these enzymes exhibit a set of conserved structural motifs that bind various cofactors, creating an intricate

wiring system for efficient electron transport⁶. A notable challenge arises when electrons need to be transported between a hydrosoluble environment and a plasma membrane.

In prokaryotes, one such family of redox enzymes addressing this challenge is the molybdenum-tungsten *bispyranopterin guanine dinucleotide (Mo/W-bisPGD) formate dehydrogenases (FDHs)*⁷,⁸. The catalytic domain, conserved through the Mo/W-PGD enzyme super-family, is sometimes fused to an additional electron-connecting module also found in hydrogenases and complex I (ref. 7). This is the case

<footnote n="1">¹Aix Marseille Univ, CNRS, LCB, IMM, IM2B, Marseille, France. ²Univ. Grenoble-Alpes, CEA, CNRS, Metalloproteins Unit, Grenoble, France. ³Aix Marseille Univ, CNRS, BIP, IMM, IM2B, Marseille, France. ⁴Aix Marseille Univ, CNRS, CEA, BIAM, IM2B, Saint Paul-Lez-Durance, France. ⁵Synchrotron SOLEIL, Saint-Aubin, France. ⁶Univ. Grenoble-Alpes, CNRS, Grenoble INP, TIMC, Grenoble, France. ⁷Univ. Grenoble-Alpes, CEA, CNRS, Electron Microscopy and Methods Unit, Grenoble, France. ⁸Aix Marseille Univ, CNRS, AFMB, IM2B, Marseille, France. ⁹Present address: Stress Adaptation and Metabolism Unit, Department of Microbiology, Pasteur Institute, CNRS, Microbiologie Intégrative et Moléculaire, Paris, France. ¹⁰These authors contributed equally: M. Broc, M. V. Cherrier. ✉e-mail: yvain.nicolet@ibs.fr; magalon@imm.cnrs.fr; annewalburger@univ-amu.fr</footnote>
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<figure><img src="image_6.png" /><figcaption>**Fig. 1 | BsForCE1 structure.** a, Cartoon representation of the full ForC~4~–ForE~4~ complex obtained by XRC. One monomer of ForC and one monomer of ForE are highlighted in dark blue and orange, respectively. The other ForC and ForE subunits of the complex are colored in light blue and yellow, respectively.
b, Crystallographic structure of one monomer of ForC in cartoon representation with the V-shape motif binding the ForE tetramer. MK-7, iron–sulfur clusters and the active site *bis*PGD coordinating the molybdenum ion are shown in ball-and-stick representation with carbon, oxygen, nitrogen, sulfur, iron and molybdenum atoms colored in light blue, red, blue, yellow, brown and turquoise, respectively.
c, Crystallographic structure of one ForE subunit in cartoon representation.</figcaption></figure>

The visible part of HMP is indicated. d, Sagittal section of ForCE showing the presence of lipids inside the cavity created by ForE tetramerization. Cryo-EM models of ForC and ForE are shown in surface representation and colored in dark blue and orange, respectively. The remaining cryo-EM Coulomb potential densities that are not explained by protein atoms are displayed in semitransparent beige surfaces contoured at 3σ. Lipids modeled in these densities are displayed in stick representation with carbon, oxygen and nitrogen atoms colored in light blue, red and blue, respectively. e, Surface representation of the ForE tetramer colored in different shades of orange and of one ForC monomer in blue semitransparent surface representation.

for the noncanonical FDHs from *Bacillus subtilis*, the two isoenzymes ForCE1 and ForCE2 (ref. 9), in which the catalytic subunit, ForC, binds six metal-containing cofactors. Electrons are wired from formate to quinone but the mechanism by which the cytoplasmic enzyme interacts with the membrane, where the quinone pool is located, remains unclear. Remarkably, ForC forms a phylogenetic clade, which is distinct from other FDHs⁹. Moreover, it is always found associated with ForE, with which it forms a complex. ForE is the only partner subunit among FDHs that lacks the signatures for a cofactor-binding motif (Extended Data Fig. 1a) but instead contains a conserved domain of unknown function (DUF1641)⁹. Therefore, we hypothesized that ForE allows connection between the cytoplasmic catalytic subunits and the membrane quinone pool. Here, we report the structural and biochemical bases of formate:menaquinone (MK) oxidoreduction in *B. subtilis*. We demonstrate that the DUF1641 found in ForE is indeed responsible for linking the FDH to the membrane. We also discovered that the genes encoding the DUF1641 domain always match with a cytoplasmic redox enzyme. Collectively, our findings reveal that this domain corresponds to a conserved structural unit for constructing oxidoreductases in prokaryotes, enabling quinone channeling between the membrane

and various redox catalytic subunits. Therefore, we propose to name
this DUF1641 the helical membrane plugin (HMP).

Results

We purified the ForCE1 complex from *B. subtilis* (*Bs*ForCE1) and determined its structure using X-ray crystallography (XRC) at a resolution of 2.69 Å and single-particle cryo-electron microscopy (cryo-EM) at 3.35 Å (Fig. 1 and Tables 1 and 2). Both methods led to almost identical structures, yet highlighting unique and highly complementary features. The overall ForCE structure corresponds to a ForC~4~E~4~ hetero-octamer, consistent with the molecular weight of 512 kDa determined by size-exclusion chromatography combined with multiangle light scattering (SEC–MALS) analysis (Extended Data Fig. 2a). The quaternary structure recalls a four-flower bunch with dimensions of about 175 × 175 × 135 Å (Fig. 1a). The four ForE subunits are exclusively constituted of α-helical segments and are intertwined into a left-handed superhelical quaternary structure, onto which the four ForC subunits are bound. Local disruption of the ForE tetramer creates a wider vestibule with four openings on the sides, sealed by a V-shape motif formed by two consecutive α-helices of each ForC subunit (Fig. 1b–e
<table><caption>**Table 1 | X-ray crystallography data collection and refinement statistics (molecular replacement)**</caption><thead><tr><th></th><th colspan="2">WT</th><th>ΔHMP</th></tr><tr><th></th><th>XDS</th><th>STARANISO</th><th>Aimless</th></tr></thead><tbody><tr><td><strong>Data collection</strong></td><td></td><td></td><td></td></tr><tr><td>Space group</td><td>F222</td><td></td><td>I422</td></tr><tr><td>Cell dimensions</td><td></td><td></td><td></td></tr><tr><td>a, b, c (Å)</td><td>210.8, 210.5, 494.2</td><td></td><td>215.9, 215.9, 215.5</td></tr><tr><td>α, β, γ (°)</td><td>90.00, 90.00, 90.00</td><td></td><td>90.00, 90.00, 90.00</td></tr><tr><td>Protein molecules per asymmetric unit</td><td>2</td><td></td><td>1</td></tr><tr><td>Resolution (Å)</td><td>49.50–3.30</td><td>49.50–2.69</td><td>150.0–3.63</td></tr><tr><td></td><td>(3.40–3.30) *</td><td>(2.99–2.69)</td><td>(3.85–3.63)</td></tr><tr><td>R<sub>merge</sub></td><td>27.6 (91.6)</td><td>28.7 (86.0)</td><td>13.9 (137.7)</td></tr><tr><td>R<sub>meas</sub></td><td>29.6 (97.8)</td><td>30.8 (92.4)</td><td>16.3 (163.0)</td></tr><tr><td>I/σI</td><td>6.2 (2.3)</td><td>6.1 (2.2)</td><td>8.6 (1.5)</td></tr><tr><td>CC<sub>1/2</sub></td><td>98.5 (85.8)</td><td>98.4 (73.7)</td><td>99.7 (58.9)</td></tr><tr><td>Completeness spherical (%)</td><td>99.9 (100.0)</td><td>56.9 (10.5)</td><td>99.6 (98.5)</td></tr><tr><td>Completeness ellipsoidal (%)</td><td>/</td><td>93.0 (65.7)</td><td>/</td></tr><tr><td>Redundancy</td><td>7.8 (8.2)</td><td>7.7 (7.4)</td><td>6.9 (6.7)</td></tr><tr><td><strong>Refinement</strong></td><td></td><td></td><td></td></tr><tr><td>Resolution (Å)</td><td></td><td>2.69</td><td>3.63</td></tr><tr><td>No. reflections</td><td></td><td>665,298 (31,920)</td><td>201,522 (30,619)</td></tr><tr><td>R<sub>work</sub>/R<sub>free</sub></td><td></td><td>19.26/23.02</td><td>28.0/31.9</td></tr><tr><td>No. atoms</td><td></td><td></td><td></td></tr><tr><td>Protein</td><td></td><td>17 557</td><td>8 501</td></tr><tr><td>Ligands and ions</td><td></td><td>604</td><td>131</td></tr><tr><td>Water</td><td></td><td>687</td><td>0</td></tr><tr><td>Wilson B factor</td><td></td><td>55.0</td><td>115.6</td></tr><tr><td><strong>B factors</strong></td><td></td><td></td><td></td></tr><tr><td>Protein</td><td></td><td>59.8</td><td>159.0</td></tr><tr><td>Ligands and ions</td><td></td><td>57.3</td><td>145.9</td></tr><tr><td>Water</td><td></td><td>28.9</td><td>-</td></tr><tr><td><strong>R.m.s.d.</strong></td><td></td><td></td><td></td></tr><tr><td>Bond lengths (Å)</td><td></td><td>0.007</td><td>0.007</td></tr><tr><td>Bond angles (°)</td><td></td><td>0.93</td><td>0.91</td></tr><tr><td><strong>Ramachandran</strong></td><td></td><td></td><td></td></tr><tr><td>Favored region</td><td></td><td>95.92</td><td>88.5</td></tr><tr><td>Allowed region</td><td></td><td>3.90</td><td>9.0</td></tr><tr><td>Outliers region</td><td></td><td>0.18</td><td>2.5</td></tr></tbody></table>

and Extended Data Figs. 3a–d and 4a). The cryo-EM map also revealed small openings at the ForC–ForE interfaces. In these regions, additional features were observed, some of which were also present in the XRC electron density map, albeit to a lesser extent (Fig. 1d, Extended Data Fig. 3c,d and Supplementary Fig. 1). They were best modeled as glycerophospholipids that would act as seals to prevent solvent access to the vestibule.

The overall structure of ForC is highly similar to the corresponding catalytic subunit of FDH from *Rhodobacter capsulatus*¹⁰ or *Methylovorubrum extorquens* AM1 (ref. 11), with root-mean-square deviations

<table><caption>**Table 2 | Cryo-EM data collection, refinement and validation statistics**</caption><thead><tr><th></th><th>(EMD-19452)<br>(PDB 8RRO)</th></tr></thead><tbody><tr><td><strong>Data collection and processing</strong></td><td></td></tr><tr><td>Magnification</td><td>36 000</td></tr><tr><td>Voltage (kV)</td><td>200</td></tr><tr><td>Electron exposure (e<sup>-</sup> per Å<sup>2</sup>)</td><td>40</td></tr><tr><td>Defocus range (μm)</td><td>-0.5 to -3.5</td></tr><tr><td>Pixel size (Å)</td><td>1.1</td></tr><tr><td>Symmetry imposed</td><td>C<sub>4</sub></td></tr><tr><td>Initial particle images (no.)</td><td>2,825,725</td></tr><tr><td>Final particle images (no.)</td><td>112,095</td></tr><tr><td>Map resolution (Å)</td><td>3.35</td></tr><tr><td>FSC threshold</td><td>0.143</td></tr><tr><td>Map resolution range (Å)</td><td>2.95–10.00</td></tr><tr><td><strong>Refinement</strong></td><td></td></tr><tr><td>Initial model used (PDB code)</td><td>8RQZ</td></tr><tr><td>Model resolution (Å)</td><td>3.2</td></tr><tr><td>FSC threshold</td><td>0.143</td></tr><tr><td>Model resolution range (Å)</td><td>2.95–10.00</td></tr><tr><td>Map sharpening B factor (Å<sup>2</sup>)</td><td>-119.5</td></tr><tr><td>Model composition</td><td></td></tr><tr><td>Nonhydrogen atoms</td><td>18,937</td></tr><tr><td>Protein residues</td><td>4,492</td></tr><tr><td>Ligands</td><td>92</td></tr><tr><td>Water</td><td>84</td></tr><tr><td><strong>B factors (Å<sup>2</sup>)</strong></td><td></td></tr><tr><td>Protein</td><td>97.95</td></tr><tr><td>Ligand</td><td>92.28</td></tr><tr><td>Water</td><td>80.14</td></tr><tr><td><strong>R.m.s.d.</strong></td><td></td></tr><tr><td>Bond lengths (Å)</td><td>0.005</td></tr><tr><td>Bond angles (°)</td><td>1.074</td></tr><tr><td><strong>Validation</strong></td><td></td></tr><tr><td>MolProbity score</td><td>2.05</td></tr><tr><td>Clashscore</td><td>22.78</td></tr><tr><td>Poor rotamers (%)</td><td>0.62</td></tr><tr><td><strong>Ramachandran plot</strong></td><td></td></tr><tr><td>Favored (%)</td><td>96.78</td></tr><tr><td>Allowed (%)</td><td>3.22</td></tr><tr><td>Disallowed (%)</td><td>0</td></tr><tr><td><strong>Model vs. Data</strong></td><td></td></tr><tr><td>CC (mask)</td><td>0.84</td></tr><tr><td>CC (main chain)</td><td>0.83</td></tr><tr><td>Mean CC for ligands</td><td>0.69</td></tr></tbody></table>

(r.m.s.d.) of 1.13 and 1.42 Å, respectively (Extended Data Fig. 5a). The N-terminal domain defines a peculiar electron-connecting module¹², which hosts three [4Fe–4S] and one [2Fe–2S] clusters (Fig. 1b and Supplementary Fig. 2) and shows remarkable structural similarities (r.m.s.d. ranging between 1.2 and 1.79 Å) to the *Clostridium pasteurianum* FeFe-hydrogenase¹³, *Thermus thermophilus* reduced

<footnote>*Values in parentheses are for the highest-resolution shell.</footnote>
a

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b

<table><thead><tr><th></th><th>g tensor characteristics<sup>a</sup></th><th>Midpoint Redox potential (mV)<sup>b</sup></th></tr></thead><tbody><tr><td rowspan="2">Fe/S<sub>1</sub> [4Fe-4S]</td><td>g<sub>1,2,3</sub> = 2.065, 1.889, 1.820</td><td>-235 (pH 6.0)</td></tr><tr><td>g<sub>av</sub> = 1.925, Δg = 0.245</td><td>-290 (pH 7.5)</td></tr><tr><td rowspan="2">Proximal [4Fe-4S]</td><td>g<sub>1,2,3</sub> = 2.044, 1.944, 1.893</td><td>-288 (pH 6.0)</td></tr><tr><td>g<sub>av</sub> = 1.960, Δg = 0.151</td><td>-351 (pH 7.5)</td></tr><tr><td rowspan="2">Fe/S<sub>2</sub> [4Fe-4S]</td><td>g<sub>1,2,3</sub> = 1.996, 1.965, 1.88-1.90</td><td>-379 (pH 7.5)</td></tr><tr><td>g<sub>av</sub> = 1.95, Δg = 0.11</td><td>-379 (pH 7.5)</td></tr><tr><td rowspan="2">[2Fe-2S]</td><td>g<sub>||,⊥</sub> = 2.018, 1.937</td><td>-426 (pH 7.5)</td></tr><tr><td>g<sub>av</sub> = 1.964, Δg = 0.081</td><td>-426 (pH 7.5)</td></tr><tr><td>S = 3/2 proximal [4Fe-4S]</td><td>g<sub>eff 1</sub> = 5.45 ± 0.01, g<sub>eff 2</sub> = 5.19 ± 0.01</td><td>-275 ± 20 (pH 6.0)</td></tr></tbody></table>

**Fig. 2** | ForC electron transfer chain. **a**, Distances among MK-7, [Fe–S] clusters and the Mo-bisPGD in one ForC subunit. MK-7, [Fe–S] clusters and the active site are shown in ball-and-stick representation with carbon, oxygen, nitrogen, sulfur, iron and molybdenum atoms colored in light blue, red, blue, yellow, brown and turquoise, respectively. The midpoint potential of MK-7 was determined previously⁹. **b**, EPR g values and redox potentials (mV versus standard hydrogen electrode) for [Fe–S] clusters of BsForCE1. EPR-monitored redox titration experiments were carried out at two different pH values (that is, 6.0 and 7.5) (Extended Data Fig. 6 and Supplementary Note 1). The proximal [4Fe–4S] most probably presents a mixture of S = 1/2 and S = 3/2 spin states (Supplementary Note 2).⁴ Average g value g<sub>av</sub> = 1/3 (g<sub>1</sub> + g<sub>2</sub> + g<sub>3</sub>) or g<sub>av</sub> = 1/3 (g<sub>||</sub> + 2 g<sub>⊥</sub>); g-tensor anisotropy Δg = g<sub>1</sub> – g<sub>3</sub>; uncertainty on g values is typically ±0.0005, unless otherwise specified; g<sub>eff</sub> is an effective g value.⁶ Uncertainty is typically ±10 mV, unless otherwise specified.

nicotinamide adenine dinucleotide (NADH): quinone oxidoreductase, complex I Nqo3 subunit¹⁴, *Hydrogenophilus thermoluteolus* NAD⁺-reducing [NiFe]-hydrogenase HoxU subunit¹⁵ and *Acetomicrobium mobile* electron bifurcating [NiFe]-hydrogenase HydA subunit¹⁶ (Supplementary Fig. 3). The C-terminal domain harbors a fifth [4Fe–4S] cluster (proximal in Fig. 1b,e and Supplementary Fig. 2) and the Mo-bisPGD cofactor, which serves as the site of formate oxidation. Strikingly, despite an overall similar fold with other FDHs, phylogenetic

analyses indicate that various substitutions in this domain make ForC unique when compared to other FDHs (Extended Data Fig. 1b). One of them, the Q392 residue, whose equivalent position corresponds to a histidine residue in all other FDHs⁹, is located at a maximal distance of 4.6 Å from the Mo atom and 9.5 Å from R585 (Extended Data Fig. 5b). While these two residues are markers of FDH activity, their exact roles are unknown. Molybdenum is coordinated by the two dithiolene groups of PGD, the conserved C391 residue and a sulfur ligand (Extended Data Fig. 5b and Supplementary Fig. 4). The ForC His-coordinated [4Fe–4S] cluster faces the vestibule window behind the V-shape motif (Fig. 1b,e and Extended Data Fig. 4a). Additional density observed near this cluster in each ForC subunit was best modeled as an MK-7 molecule. Remarkably, it has full occupancy in both the crystal and the cryo-EM structures (Fig. 1d,e, Extended Data Fig. 3a,b and Supplementary Fig. 4), which is consistent with previous work⁹. Although the hydrophobic tail of MK-7 cannot be entirely modeled because of partial disorder in XRC, the EM density indicates that all four MK-7 molecules point toward the vestibule and interact with other lipids inside (Fig. 1d,e, Extended Data Fig. 3a,b and Supplementary Fig. 1). The MK-7 ring is inserted between two α-helices (residues 223–236 and 240–257) of ForC with a V-shape motif, which defines an opening connecting the MK ring to the His-coordinated [4Fe–4S] cluster at a distance of 8.1 Å (Fig. 1b and Extended Data Fig. 4a). Both α-helices are amphipathic, orienting conserved polar residues toward the head of MK-7. For instance, the oxygen atom O1 establishes hydrogen bonds with the carboxyl group of ForC E237 residue and a water molecule, while atom O4 forms hydrogen bonds with the main chain amide and the Oy1 atoms of the ForC T100 residue (Extended Data Fig. 4a and Supplementary Fig. 4). The remaining surface of these helices display hydrophobic residues that interact with the ForE subunit.

The average distance between redox centers in the BsForCE1 structures falls within the range of 8.1–18.5 Å, which is compatible with rapid electron transfer from formate to MK-7 (Fig. 2a). Electron paramagnetic resonance (EPR)-monitored redox titration experiments enabled us to determine the signature and redox potential of four of the five [Fe–S] centers and to assign the [4Fe–4S] cluster located near the Mo active site (that is, the proximal [4Fe–4S]) and the [2Fe–2S] cluster (Fig. 2, Extended Data Table 1, Extended Data Fig. 6 and Supplementary Note 4). The proximal [4Fe–4S], the [2Fe–2S] and the unassigned [4Fe–4S] Fe/S<sub>2</sub> clusters are nearly isopotential, with values ranging between –350 and –426 mV (Fig. 2 and Extended Data Fig. 6). The midpoint potential of the [2Fe–2S] cluster in BsForCE1 (–426 mV; Fig. 2) is substantially lower than that measured in the *Cupriavidus necator* FDH (–305 mV)¹⁷ or the *R. capsulatus* FDH (–264 mV at pH 9.0)¹⁸. Interestingly, this cluster is not the final electronic relay in BsForCE1 as it is too far from the quinone-binding site (23.6 Å; Fig. 2a) in contrast to other FDHs¹⁰,¹¹. This cluster is strictly conserved in all ForC sequences; however, its exact role remains to be determined (Extended Data Fig. 1a).

The structure of the ForE tetramer defines a 95-Å-long square tube with side and diagonal dimensions of about 20 and 30 Å, respectively. The N-terminal end of the ForE tetramer can be best described as a frayed braided rope (Fig. 1e). Residues 2–14 correspond to the four frayed strands, each intertwining with one ForC subunit, probably preventing unintended motions and adding rigidity to the whole ForC<sub>4</sub>E<sub>4</sub> structure (Fig. 1c,e). Helices 1–3 of the four ForE subunits delineate the N-terminal end of the tube. Helix 4 from each ForE monomer introduces a break in this superhelical structure and defines a broader vestibule with four windows where the four ForC subunits are plugged (Fig. 1c–e and Extended Data Fig. 3a,b). Helices 5–8 correspond to the second intertwined superhelical region that defines the second part of the tube. At the C terminus, residues 120–155 correspond to DUF1641. The first seven residues of this domain are missing in both the XRC and the cryo-EM models, most likely because of disorder. Yet, the other residues are clearly visible in both structures, where they define α-helices
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 3 | ForCE is connected to the membrane quinone pool through HMP.**
a, ForCE1 and ForCE2 are part of the aerobic respiratory chain. WT, ΔforCI, ΔforC2 and ΔforCIΔforC2 cells were cultured with formate and ΔforC2 transformed with pForCE1 with (+) or without (−) 0.8% xylose as an inducer. b, HMP connects ForCE to the respiratory chain. The ΔforC2ΔforE1 strains transformed with pForCE1, pForCE1AHMP and pForCE1f128D-R132D were grown with (+) or without (−) 0.8% xylose. For each strain, O₂ consumption was measured using an oxygen electrode before (green dots) and after (blue dots) the addition of formate in the O₂-saturated buffer. Each dot represents a replicate and, for each condition, the min, max, median and first and third quartile values are shown (\*\*P = 0.0047 (ΔforCI) and 0.0059 (ΔforC2 pForCE1') and \*\*\*\*P < 0.0001; NS, not significant). Statistical analysis with *n* ≥ 3 biological replicates was performed using a two-way ANOVA with Šidák correction for multiple comparisons.</figcaption></figure>

7 and 8. Interestingly, to end the tube, helix 8 does not follow the same superhelical structure but instead loops back into the tube mesh below helix 7, resembling a knit stitch (Fig. 1c, Extended Data Fig. 7 and Supplementary Fig. 5). This likely contributes to locking and stabilizing the tube structure. After this module, the last nonconserved residues (158–186), predicted to be intrinsically disordered, are not observed in both structures. This ForE structure is highly reminiscent of the one of the HucM subunit of the [NiFe] hydrogenase from *Mycobacterium smegmatis*¹⁹. In the latter, four HucM subunits also define a large superhelical tubular structure, onto which eight catalytic subunits (four dimers of HucSL protomer) are bound through an amphipathic helix provided by HucS (Extended Data Fig. 4). The HucM structure was predicted to interact with the membrane²⁰. Yet, of the 188 amino acids of HucM, only residues 20–79 could be modeled into the cryo-EM maps.

The ForE tetramer constitutes the structural core that holds the whole BsForCE1 structure, while the four ForC subunits do not interact with each other (Fig. 1a). Strikingly, whereas the external surface of the tube is hydrophilic, as can be seen in Extended Data Fig. 3e, the inside of the tube is entirely lined up with hydrophobic residues, resulting in

a lipophilic surface throughout its entire length. In both the XRC and the cryo-EM experimental maps, the N-terminal end of the tube is not empty; instead, it is filled with long features that were best modeled as four aligned fatty acids (Fig. 1d and Extended Data Fig. 7). Strikingly, the lipophilic tails point inward the tube, while the carboxyl groups are located at the entrance, pointing toward the surface. This configuration, reminiscent of lipid monolayers, likely acts as a barrier to prevent solvent access inside the tube. Remarkably, in the cryo-EM map, the C-terminal end of the tube is capped by elongated features that were also modeled as additional fatty acids, (Fig. 1d and Extended Data Fig. 3). The lack of such lipid cap in the XRC structure most likely results from crystal packing contacts in this region that induced release of the lipids (Extended Data Fig. 7). The outward-facing α7 helices at the tube terminus consist of hydrophobic and basic residues, thus facilitating interaction between these lipids and the protein (Extended Data Figs. 2f, 3e and 7). Therefore, this unique structural arrangement combined with the presence of a lipid layer suggests that the DUF1641 function is to connect the FDH to the membrane and to provide access to the membrane MK pool. Therefore, we propose to rename it HMP.

We next probed whether ForCE is connected to the membrane quinone pool in a respiratory electron transfer chain in *B. subtilis*. Noteworthy, *B. subtilis* contains two copies of the *forCE* gene tandem, named *forCE1* and *forCE2* (ref. 9). The ForC sequences share 61% of identity and both ForE1 and ForE2 contain a highly similar HMP domain. Oxygen consumption of a *B. subtilis* cells suspension was monitored using an oxygen electrode following sequential injection of formate into an O₂-saturated buffer (Fig. 3a). Formate addition led to a significant increase in the O₂ consumption rate in the wild-type (WT) strain, demonstrating that electrons derived from formate oxidation indeed contribute to the reduction of O₂. Furthermore, this behavior was almost abolished upon addition of KCN or HQNO, common inhibitors of oxygen reductases and quinone-reacting enzymes, respectively (Extended Data Fig. 2b,c). Moreover, there was no increase in the rate of O₂ consumption in the ΔforC2 and the ΔforC1ΔforC2 strains, demonstrating that, under these conditions, formate oxidation and O₂ consumption are mainly coupled by ForCE2. Next, to ensure *forCE1* overexpression, an artificial inducible promoter was used. As expected, in the absence of *forC2* and the inducer (*pForCE1*⁻), there was no increase in O₂ consumption. However, in the presence of the inducer (*pForCE1*⁺), the addition of formate led to a twofold increase in the rate of O₂ consumption, highlighting that ForCE1 can supplement the lack of ForCE2 in coupling formate oxidation to O₂ reduction (Fig. 3a). We further assessed the role of HMP, by either completely removing it (ΔHMP) or altering the helix 7 amphipathic characteristic (F128D;R132D double substitution). In both cases, the addition of formate did not result in any change in the rate of oxygen consumption, despite the production of a stable and active complex (Fig. 3b, Extended Data Fig. 2a,d,e,g and Supplementary Table 1). This demonstrates the key role of HMP in coupling formate oxidation catalyzed by the ForC subunit to the oxygen consumption taking place in the membrane. This observation, combined with our structural data, strongly supports that HMP, because of its amphipathic character, is responsible for the interaction of the ForCE complex with the membrane.

We next explored the diversity of the HMP (DUF1641)-containing protein family, which represents 4,092 sequences from bacterial and archeal phyla. Sequence similarity network (SSN) and genome neighborhood network (GNN) analyses allow pinpointing possible relationships among evolutionarily related proteins, considering their genomic context (Fig. 4a). Our analysis revealed four distinct genomic arrangements, labeled types I to IV. In all of them, the oxidoreductase-encoding gene is found in synteny with another gene that contains a sequence encoding HMP. These oxidoreductases are predicted to catalyze low-redox-potential reactions and all share quinone as the electron acceptor. Type I is the most common arrangement, which features a gene encoding a molybdoenzyme upstream of the HMP-encoding gene
<figure><img src="image_10.png" /><figcaption>**Fig. 4** | SSN and GNN analyses of the HMP (DUF1641) family identify four types of functional arrangements. **a**, SSN of the Interpro IPR012440 (DUF1641) family of sequences using a threshold of 30. The main clusters are numbered on the basis of the number of nodes or sequences they group and are color-coded according to the gene synteny. Cluster 6 has synteny indicating that it belongs to the type II organization but has an MSA consistent with a SQR family (type III). GNN along with MSA predicted four types of functional arrangements (labeled types I to IV) showing synteny with a molybdoenzyme (I), an NDH2 (II), an SQR</figcaption></figure>

(III) or a two-subunit hydrogenase (IV). **b**, Conserved structural unit to build four quinone oxidoreductases: FDH ForCE (this work), NDH2 (AlphaFold prediction), SQR (AlphaFold prediction) and the hydrogenase Huc structure¹⁹. These diverse bioenergetic systems use a tube-like structure to connect the membrane through the HMP domain (colored in red) with the catalytic subunits (represented as transparent cartoons). Overall, these arrangements allow the long-distance connection of quinones with different redox centers.

and is best illustrated by the ForCE enzymes from *B. subtilis*. The type II arrangement corresponds to the gene encoding the NADH:quinone oxidoreductase (NDH2)²¹ located downstream of the HMP gene. The type III cluster gathers a sequence encoding a sulfide:quinone oxidoreductase (SQR)²² upstream of the HMP gene. The type IV arrangement is exemplified by the *hucMSL* genetic organization and features genes encoding [NiFe]-hydrogenase¹⁹. In each arrangement, different patterns of clustering suggest divergent evolution, which is further supported by a separate clustering of archaeal and bacterial systems in arrangements I, III and IV (Extended Data Fig. 8). Lastly, we observed that the sequence length of the HMP subunit is also an important factor in the clustering, as sequences in a given cluster tend to have a homogeneous size (Extended Data Fig. 8).

Discussion

HMP is a strategy to overcome the challenge of transporting electrons between a hydrosoluble compartment and the membrane quinone pool. This module allows various oxidoreductases to interact with the membrane and to access the quinone pool. In this work, this mechanism has been thoroughly studied using the noncanonical FDH, the ForCE enzyme. Remarkably, among the wide family of Mo/W-bisPGD enzymes, ForCE is the first example of an enzyme with such a structural module to connect the membrane quinone pool. In the cell, ForCE is a respiratory complex that couples formate oxidation with oxygen consumption, indirectly participating in the generation of

the proton-motive force through oxygen reductases²³. Thus, ForCE emerges as a full-fledged player in the respiratory chain of *B. subtilis*. The small ForE subunit possesses a distinctive quaternary structure characterized by entanglement of the four chains, forming a 95-Å-long helical hydrophobic tube with four openings on the sides, each facing a ForC subunit. The quinone-binding site is delineated by a pocket within ForC where two conserved residues interact with the quinone ring while the hydrophobic chain extends toward the internal tube. A similar mode of interaction is observed in the hydrogenase Huc at the HucS-HucM interface. In contrast to the V-shape motif, specific to ForC (Methods), that seals the ForE vestibule, two HucS subunits close the HucM opening, each contributing through an α-helix (Extended Data Fig. 4). Apart from the HMP domains, HucM and ForE do not share extensive primary sequence identity. However, they both form a super-helical structure likely defining a conduit for transporting lipophilic MKs over a long distance (60 Å for ForCE1 and 94 Å for Huc) between the cytoplasmic catalytic subunit(s) and the membrane. This is supported by the internal dimensions (16–38 Å for the ForE tube) and hydrophobic characteristic of both tubes. Notably, the detection of lipids inside the ForE conduit suggests that this tube is never solvated, enabling quinones to enter or exit the tube without notable energy barriers. At the top of the tube is the HMP structural module responsible for the interaction between ForCE and the membrane. HMP is, in fact, a small structural motif with a distinctive fold, widely found in bacteria and archaea. It exhibits an amphipathic profile, enabling it to interact
with both hydrophobic and aqueous environments. Furthermore, the positively charged surface supports transient interactions between HMP and the negatively charged heads of the lipids at the membrane. Hence, it excludes a deep anchoring of the ForCE (and likely Huc) into the membrane, in agreement with the purification of both enzymes in the soluble fraction. Deletion of the HMP domain does not result in disruption of the complex ForCE, highlighting that its quaternary structure does not depend on the HMP domain. In contrast, while FDH activity is maintained, formate oxidation is no longer coupled to O₂ reduction, demonstrating the key role of the HMP module in connecting the two reactions. Taken together, our findings underscore the unique role of HMP to facilitate transient interactions of cytoplasmic oxidoreductases with the membrane. The sequence encoding HMP is mainly observed in genes associated with oxidoreductases such as FDH, [NiFe]-hydrogenase, NDH2 or SQR. Evolution has repeatedly used HMP to build several enzymes that share the same function, that is, to connect oxidoreductases to the membrane quinone pool (Fig. 4b). This plugin enables these enzymes to use the reducing power generated at their catalytic subunits to reduce quinones and, thus, converts them into quinone reductases. While four quinones reductases were identified in this work, such a plugin capping a hydrophobic tube might also channel quinols to cytoplasmic oxidases. The identification of HMP increases the repertoire of the potential interconnections between redox processes in bioenergetic pathways. The existence of HMP suggests that other yet unidentified structural domains may soon be added to the list of interaction modules between proteins or between cellular compartments. Such domains allow for greater integration of different metabolic pathways contributing to the functioning and adaptation of organisms to their environment. We can envision harnessing the unique qualities of the HMP domain for biotechnological innovations, taking advantage of the plug-and-play characteristic of HMP to connect function to different compartments.

## Online content

Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41594-025-01607-4.

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# Methods

## SSN-GNN analysis

To compute an SSN, we used the InterPro family IPR012440 as an entry to the Enzyme Function Initiative (EFI) enzyme similarity tool interface, with a threshold of 30, which corresponds to the recommended value observed at 35–40% pairwise identity within the family. The resulting network was visualized using Cytoscape and the organic layout. We then used the GNN analysis tool in conjunction with multiple-sequence alignments (MSAs) to evaluate the 30 most important clusters (comprising over 85% of the nodes).

## MSA analysis for type II and III arrangements

In type II and III arrangements, the gene located downstream and upstream of the HMP gene, respectively, encodes a pyridine nucleotide disulfide oxidoreductase (Pyr_redox_2 family). Members of this family catalyze various types of reactions and are characterized by two Rossmann fold domains, which bind FAD and a pyridine nucleotide or hydrogen sulfide in the case of the sulfide dehydrogenase family²⁴. To understand the differences between type II and type III arrangements, we collected representative sequences from each cluster (167 sequences (Supplementary Table 2) with distinct genera in clusters 2, 4–6, 8–11, 14, 16, 18–19, 22 and 25) and performed sequence alignments using Clustal Omega from (https://toolkit.tuebingen.mpg.de/tools/clustalo) with default parameters. Type II corresponds to sequences that contain FAD, NADH and quinone-binding consensus motifs defining NDH2 (ref. 21). In contrast, type III gathers sequences that lack the NADH-binding motif but harbors a two-cysteine motif usually found in SQR²².

## Crystallization, data collection and structure determination

Crystallization of *BsForCE1* was performed under anaerobic conditions inside a glovebox following the procedure described previously²⁵. Sitting drops were prepared by mixing 1 µl of a 30 mg ml⁻¹ protein solution with 1 µl of 1.5 M sodium malonate pH 7 reservoir solution and equilibrating in the vapor phase against 1 ml of the latter. Reproducible crystals appeared after a few days. The crystals were cryoprotected using 2 M sodium malonate pH 7 and 30% glycerol and were subsequently flash-cooled in liquid propane inside the chamber²⁵. *BsForCE1-ΔHMP* was concentrated to 10 mg ml⁻¹ and crystals were obtained by mixing 0.7 µl of protein with 0.7 µl of a solution made of 2.1 M ammonium sulfate and 1.15 M triammonium citrate. Crystals were cryoprotected in the same mother liquor composition supplemented by 30% glycerol and flash-cooled in liquid nitrogen before data collection. X-ray diffraction data were collected at beamline ID23-2 of the European Synchrotron Radiation Facility (ESRF). Data indexation was carried out using XDS²⁶ and, because of the high anisotropy (Extended Data Fig. 9 and Supplementary Figs. 8 and 9), subsequently with STARANISO (Table 1)²⁷. The data from the ForCE-ΔHMP were indexed with XDS and scaled with Aimless from the CCP4 suite²⁸. The crystal structure was determined by molecular replacement using a previously built in-house model and Phaser software²⁹. Crystallographic refinement was conducted using *Buster*³⁰ and the three-dimensional model was examined and modified with *Coot*³¹. The atomic coordinates of the final model and diffraction data were deposited to the Protein Data Bank (PDB 8RQZ for *BsForCE1* and PDB 9GZQ for *BsForCE1-ΔHMP*).

## Cryo-EM grid preparation under anaerobic conditions

Cryo-EM grids (QUANTIFOIL grids Cu/Rh 300-mesh, R 2/1) were glow-discharged for 45 s at 30 mA under low vacuum before being introduced in a custom-made glovebox (JACOMEX) housing a Vitrobot Mark IV freezing system (FEI). To prepare the *BsForCE1* cryo-EM grids under anaerobic conditions, we used the same protocol as was previously described³⁰. Then, 4 µl of protein solution at 0.3 mg ml⁻¹ was applied to the grid, which was then blotted and flash-cooled in liquid propane. The Vitrobot was set to operate at 22 °C and 100% humidity

(blotting time, 2 s; blot force, 1). The same protein sample batch as that used for the crystallization experiments was used to prepare the grids, without any further processing.

## Cryo-EM data acquisition and image processing

*BsForCE1* data were collected on a Glacios cryo-EM instrument (Thermo Fisher Scientific) operating at 200 kV, at a nominal magnification of ×36,000 (corresponding to 1.1 Å per pixel at the specimen level), with a defocus range of −0.5 to −3.5 µm. Videos were recorded using a K2 summit (Gatan) direct electron detector operating in counting mode with the SerialEM software³². A series of 4,977 40-frame videos were collected with a total dose of 40 e⁻ per Å² (Table 2).

*BsForCE1* reconstruction was performed using cryoSPARC (version 3.3.2)³³. Each video was first drift-corrected (frames 2–40) for full-frame motion and sample deformation before estimating the contrast transfer function (CTF). The ForCE crystal structure was used to generate two-dimensional (2D) projections subsequently used as templates to perform an automated picking of the particles on the micrographs. A total of 2,825,725 particles were extracted and 2D sorted. The best classes, obtained from 153,476 particles, were selected and used to calculate three ab initio three-dimensional (3D) models subsequently used for a last step of particle curation using a hetero refinement job. A total of 112,095 particles were then used to calculate a first 3D reconstruction. Sharpening of the corresponding Coulomb potential density map using a *B* factor of −124.5 Å², led to a map at 3.40-Å resolution. Subsequently, refinement of the global CTF was performed and the final map was sharpened using a *B* factor value of −119.5 Å², leading to a 3.35-Å-resolution map using the Fourier shell correlation (FSC) 0.143 cutoff criterion. *C*₄ symmetry was applied in all 3D refinement steps. Finally, the local resolution of the reconstruction was estimated using cryoSPARC procedure.

## Model building in the cryo-EM maps

The *BsForCE1* crystal structure was used as a starting model and was fitted into the cryo-EM density map as a rigid body using one ForC and one ForE monomers. The tetramer was then generated using the *C*₄ symmetry observed in the complex. Several iterative cycles of manual modification using *Coot*³¹ and refinement with PHENIX²⁹ were necessary to model the complete structure into the reconstructed cryo-EM density map. For the first cycle, only one monomer of ForC and one monomer of ForE were reconstructed. Then, using *C*₄ symmetry, the full protein was refined. Residues that did not match the reconstructed cryo-EM density map were removed from the model. The water molecules that were visible in the cryo-EM map and present in the reference crystal structure were added to the model (Table 2). Density corresponding to the naphthoquinone part and the beginning of the isoprenoid chain of MK-7 was clearly visible and modeled. The rest of the isoprenoid chain was less clear, perhaps because it can adopt multiple positions; however, one possible conformation was modeled. Most of the remaining unexplained densities could be interpreted as lipids. Nevertheless, a few densities remained uninterpretable. The atomic coordinates and the corresponding cryo-EM map were deposited to the PDB and EM Data Bank (EMDB), respectively (PDB 8RRO and EMD-19452).

Figures of structures were generated using ChimeraX³⁴ and PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).

## Bioinformatic analysis of multidomain FDHs

Protein representatives of the FDH family whose enzyme function was previously elucidated by biochemical or structural methods⁹,³⁵⁻³⁸ were used as bait for BLAST queries to construct a comprehensive library of sequences, filtered to include an amino-terminal electron-connecting module. When sequences were more than 90% identical, only one sequence was retained. As such, a total of 442 sequences were obtained (Supplementary Table 3) and further
analyzed as detailed below. The sequences were aligned using the ClustalW module from Geneious 10.2.6 (https://www.geneious.com/) with default parameters. To account for the highly variable N-terminal electron-connecting module (containing multiple cofactor-binding motifs), structure-informed alignment was necessary. Essentially, available structural data from the FDHs from *R. capsulatus* (PDB 6TGA) and *M. extorquens* AM1 (PDB 7VW6) were enriched with AlphaFold2 model predictions³⁹ of distinct representatives of the dataset and structurally aligned by PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger). The MSA of the N-terminal region was manually adjusted on the basis of the structural alignments described above. The resulting alignment was further processed to extract the alignment corresponding to the conserved C-terminal domain (trimmed 2 aa before the first coordinating cysteine residue of the iron-sulfur cluster-binding motif and containing the Mo/W-bisPGD cofactor-binding motif) and that corresponding to the highly variable N-terminal domain. Each of the three alignments was then used to construct a maximum-likelihood phylogenetic tree using the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at/)⁴⁰ with automatic best-fit substitution model selection⁴¹; the model tree was refined using ultrafast bootstrap and 1,000 replicates⁴². Tree representations were prepared using iTOL v6 (https://itol.embl.de/)⁴³ and manually annotated.

To determine that the V-shape motif is specific to ForC sequences, the abovementioned MSA of the N-terminal domain was trimmed to only consider the sequence corresponding to the structural motif defining the interaction of ForC with ForE and subsequently used with the Skyline program (http://skylign.org ref. 44). The obtained hidden Markov model protein profile was then used with the HMMER program⁴⁵ to retrieve sequences from publicly available protein sequence databases (reference proteome). All retrieved sequences exclusively correspond to ForC on the basis of the following criteria: presence of the motifs allowing the coordination of the five iron-sulfur clusters and the CQ-RGH motif.

## Growth conditions and purification procedures

*B. subtilis* 3610, *B. subtilis* 3610 *comf*²¹², 168 (*trpC2*) and its derivatives were grown at 37 °C, under rotary shaking at 180 rpm, in Luria-Bertani (LB)⁹, M9 (ref. 46) or *CE*⁴⁷ minimal medium. Furthermore, 12.5 mM sodium formate or 0.8% xylose (at an optical density at 600 nm (*OD*<sub>600</sub>) of ~0.3) was added when specified. When necessary, antibiotics were used at the following concentrations: spectinomycin, 100 µg ml⁻¹, kanamycin, 10 µg ml⁻¹; erythromycin, 1.25 µg ml⁻¹. For O₂ consumption experiments, after isolation on LB agar plates, cells were grown in LB medium until stationary phase. When necessary, cells were adapted to M9 medium by diluting 500-fold in fresh medium and grown overnight. Cultures in M9 or *CE* minimal medium were then inoculated at *OD*<sub>600</sub> = 0.05 with the previous M9 or LB preculture, respectively, and cultivated until midexponential phase (*OD*<sub>600</sub> ≈ 1). For overproduction of recombinant proteins, cells were cultivated according to a previous report⁹. Purification procedures were as described previously⁹ with the difference being that they were performed in the absence of oxygen in an anaerobic chamber (95.5% N₂ and 4.5% H₂) at room temperature.

## O₂ consumption measurements

O₂ consumption measurements were performed by polarography with a Clark-type electrode at 37 °C. Cells collected at *OD*<sub>600</sub> ≈ 1 were washed out three times in M9 buffer (medium devoid of glucose and formate), concentrated and added to the oxygraph chamber containing 1.8 ml of air-saturated M9 buffer. Variations of O₂ concentration were recorded before (that is, with endogenous substrates) and after the addition of 16 mM formate. When specified, 0.16–7.6 mM *MKCN* and 1.29–7.73 µM HQNO (*N*-oxo-2-heptyl-4-hydroxyquinoline) were added. An air saturation of 100% corresponded to 211.3 µM O₂ dissolved at 37 °C.

For data treatment, O₂ consumption was normalized with the total protein concentration (nmol O₂ min⁻¹ mg⁻¹), carried out by the Bradford assay supplemented with 0.1 N NaOH and using BSA as a standard. For the WT strain, 100% corresponded to 65 nmol O₂ min⁻¹ mg⁻¹. For the *ΔforC2ΔforE1* pForCE1⁺ xylose-induced strain, 100% corresponded to 85 nmol O₂ min⁻¹ mg⁻¹. Statistical analysis with at least three biological replicates was performed with GraphPad Prism software using a two-way analysis of variance (ANOVA) with Šidák correction for multiple comparisons.

## Strain construction

To generate *B. subtilis*, 3610 *ΔforC1*, *ΔforC2* and *ΔforC1ΔforC2* and *B. subtilis* 3610 WT or *ΔforC1:kan* were transduced with phage SPP1 isolated from lysates of *B. subtilis* 168 *trpC2* *ΔforC1:ery* (BKE12160), *ΔforC2:kan* (BKK27220), *ΔforC1:kan* (BKK12160) and *ΔforC2:ery* (BKE27220). To generate *B. subtilis* 168 *trpC2* *ΔforE1ΔforE2* and *B. subtilis* 3610 *comf*²¹², *ΔforC2ΔforE1*, *B. subtilis* 168 *trpC2* or 3610 *comf*²¹² was transformed with genomic DNA *ΔforE1:ery* (BKE12170), *ΔforE2:kan* (BKK27230), *ΔforC2:ery* (BKE27230) and *ΔforE1:kan* (BKK12170), according to a previous report⁴⁸. When specified, strains were then transformed with the corresponding plasmids (pForCE1, pForCE1f128D:R132D or pForCE1fHMP) or transduced with phage SPP1 isolated from lysate of *B. subtilis* 168 *trpC2* pForCE1 at the *amyE* locus. Strains used in this study are in Supplementary Table 4.

## Plasmid construction

Plasmids pForCE1fHMP and pForCE1f128D:R132D were constructed by site-directed mutagenesis using PCR. pForCE1 (ref. 9) was amplified with primers 2079 (5'-AGAAATGATGAAACAGTGAAGAAAAGACAGGTT-3') and 2080 (5'-TGTTTCATCATTTCTGCTCTACAGCGCTTGC-3') to introduce a stop codon before the gene region encoding the HMP (DUF1641) domain or with primers 2192 (5'-CAGTGAAGAAAAGACAGAGTTATGATGATATTATCGATTCGCTAAAAGACCCGGAAATC-3') and 2193 (5'-GATTTCCGGGTCTTTTAGCGAATCGATAATATCATCATA ACCTGTCTTTTCTTCACTG-3') to substitute F¹²⁸ and R¹³² codons with D codons.

## SEC-MALS analysis

Analytical SEC-MALS profiles were recorded at eight angles using a DAWN laser photometer (Wyatt Technology) and differential refractometer (Optilab, Wyatt Technology). Then, 45 µl at 5 mg ml⁻¹ of BsForCE1 WT, BsForCE1f128D:R132D and BsForCE1fHMP were applied to a Superdex 200 10/300 Increase column (Cytiva) equilibrated with 50 mM MES pH 6.5 and 150 mM NaCl at a flow rate of 0.6 ml min⁻¹. The data were analyzed using ASTRA 7.3.2.

## EPR-monitored redox titrations

Redox titrations were carried out anaerobically at room temperature (about 25 °C) either in a glovebox or in an airtight vessel flushed with oxygen-free argon. Redox potentials were measured with a combined PtAg-AgCl-KCl (3 M) microelectrode and are given in the text with respect to the standard hydrogen electrode. Redox mediators were used at 10 µM final concentrations⁹. Reductive titrations were carried out by stepwise addition of an anaerobic sodium dithionite solution. Samples were anaerobically transferred into calibrated EPR tubes that were rapidly frozen.

X-band EPR spectra were measured on a Bruker Elexsys E500 spectrometer equipped with an ER4012ST rectangular cavity fitted to an Oxford Instruments helium flow cryostat. Numerical simulations of [Fe–S] EPR spectra were performed with the EasySpin package (release 5.0.12)⁴⁹ using Matlab (MathWorks)⁴⁴. The individual contributions of magnetically isolated S = 1/2 [4Fe–4S]⁺ clusters were calculated using a g-strain anisotropic linewidth model. The full widths at half maximum of the uncorrelated Gaussian distributions of the g principal values are given in the Extended Data Table 1.
Potentiometric titration data of proximal [4Fe-4S], [2Fe-2S] and Fe/S<sub>2</sub> were analyzed using an *n* = 1 Nernst curve, while that of Fe/S<sub>1</sub> [4Fe-4S] was fitted using a simplified model of two interacting centers as described in Supplementary Note 3.

**Reporting summary**

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

**Data availability**

The atomic coordinates of the final models, diffraction data and corresponding cryo-EM maps were deposited to the PDB and EMDB under accession codes PDB 8RQZ, PDB 8RR0 and EMD-19452 for BsForCE1 and PDB 9GZQ for BsForCE1-ΔHMP. Source data are provided with this paper.

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## Acknowledgements

We are grateful to E. Foulquier-Khadaroo and A. Galinier (Aix Marseille Univ, CNRS, LCB) for providing protocols and advice for *B. subtilis* genetics and culture, C. Aubert and B. Py (Aix Marseille Univ, CNRS, LCB) for lending us the anaerobic chamber, E. Launay (Univ. Grenoble-Alpes) for her contribution to the cryo-EM analysis, G. Brandelet (Aix Marseille Univ, CNRS, CEA, BIAM) for her contribution to the XRC analysis, B. Legeret (Aix Marseille Univ, CNRS, CEA, BIAM), H. Le Guenno (Plateforme de microscopie, IMM, Aix Marseille Univ, CNRS, Marseille, France) and A. Kosta (Plateforme de microscopie, IMM, Aix Marseille Univ, CNRS, Marseille, France), the EPR facilities available at the Aix-Marseille EPR center, the financial support from the IR INFRANALYTICS FR2054 for conducting the research, the ESRF for provision of synchrotron radiation facilities under proposal number mx2398 and the local contact A. Gautam for assistance and support in using beamline ID23-eh2 (https://doi.org/10.15151/ESRF-ES-925342287). This work was supported by the French National Research Agency in the framework of the Investissements d'Avenir program (ANR-15-IDEX-02-YN) through the funding of the 'Origin of Life' project of the Univ. Grenoble-Alpes. This work used the platform PBSIM de l'AFMB supported by FRISBI and the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology, which is supported by FRISBI (ANR-10-INBS-05-02-YN) and GRAL and financed within the Univ. Grenoble-Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003-YN). The EM facility is supported by the Auvergne-Rhône-Alpes region, the Fondation pour la Recherche Médicale, the fonds FEDER and the GIS Infrastructures en Biologie Santé et Agronomie. I.B.S. acknowledges integration into the Interdisciplinary Research Institute of Grenoble. This work was also

supported by the French National Research Agency (ANR-22-CE44-0035-01-AW), the CNRS and the CNRS Energy Unit (Cellule Energie) through the project MICROBIOCO2. U.A. and B.M. were supported by an MESR fellowship and A.-C.R. was supported by an ANR MOLYERE (ANR 16-CE29-0010-01-BG) project postdoctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

## Author contributions

W.A., A.-C.R., A.P., M.A., C.M.V., B.M. and G.S. designed the experiments. U.A., W.A., P.F., S.F., G.B., B.G., L.P., B.M., A.-C.R., M.M.J., A.P., M.L., S.G., C.M.V., M.A. and G.S. performed the experiments. M.A., W.A., G.B., A.-C.R., C.M.V., N.Y., B.M., A.P. and G.S. wrote, reviewed and edited the manuscript. M.A., W.A., G.B., G.S., A.P. and N.Y. provided the resources and obtained funding.

## Competing interests

The authors declare no competing interests.

## Additional information

Extended data is available for this paper at
https://doi.org/10.1038/s41594-025-01607-4.

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s41594-025-01607-4.

**Correspondence and requests for materials** should be addressed to Y. Nicolet, A. Magalon or A. Walburger.

**Peer review information** *Nature Structural & Molecular Biology* thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Dimitris Typas, in collaboration with the *Nature Structural & Molecular Biology* team.

**Reprints and permissions information** is available at
www.nature.com/reprints.
a.

<figure><img src="image_11.png" /></figure>

b.

Tree scale: 1

<figure><img src="image_12.png" /></figure>

Extended Data Fig. 1 | See next page for caption.
**Extended Data Fig. 1** | In-depth bioinformatics analysis of cytoplasmic multidomain FDHs. a. Phylogenetic tree generated of the N-terminal domain of 442 FDH catalytic subunit sequences (Cata SU) using a maximum likelihood approach. Inter-sequence identity was reduced to a maximum of 90% between any two sequences. Sequences forming a clade have been collapsed.

The presence of specific redox centers was deduced from the identification of conserved motifs in the primary amino acid sequence and the partner subunit(s) from the genomic organization. The arrow indicates the position

at scale between the N-terminal and the C-terminal domain. Characterized representatives are indicated: *Bacillus subtilis* (1), *Methylobacterium extorquens* AM1 (2), *Rhodobacter capsulatus* (3) and *Gottschalkia acidurici* (4).

b. Phylogenetic tree of the C-terminal domain of 442 FDH sequences using a maximum likelihood approach. Each branch tip represents a single sequence. ForC sequences are colored red. Both trees were built with IQ-TREE using the WAG + I + G4 model. Supports at nodes are ultrafast bootstrap values above 95% and are indicated by a black circle. iTOL 6.8.1 was used to generate the figures.
Molar mass (g/mol)

UV

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

f.

<figure><img src="image_18.png" /></figure>

118-K N S E E K T G Y F D I I R S L K D P E I N K S I T L L F S F L K G M G Q - 155

α7

α8

g.

<figure><img src="image_19.png" /></figure>

Extended Data Fig. 2 | See next page for caption.
**Extended Data Fig. 2** | Biochemical and structural analysis of *BsForCE1* and variants. **a.** ForC and ForE form a complex with a 4:4 stoichiometry as shown by SEC-MALS analysis of *BsForCE1* WT (blue curve), *BsForCE1*<sup>F128D-R132D</sup> (green curve) and *BsForCE1*<sup>ΔHMP</sup> (orange curve). Left y-axis, molar mass; right y-axis normalized UV chromatograms, measured molar mass in coloured stars. For *BsForCE1* WT and *BsForCE1*<sup>F128D-R132D</sup>, complexes have a MW of 512 ± 1.5 and 495 ± 1.5 kDa respectively. For *BsForCE1*<sup>ΔHMP</sup>, the complex has a MW of 470 ± 1.9 kDa. In addition, two proteins of 117 ± 1.2 kDa and 111 ± 0.8 kDa could correspond to a monomer of ForC1. **b.** **c.** Inhibition of the formate:O₂ consumption by respiratory chain inhibitors. WT strain was grown with 12.5 mM formate. O₂ consumption was measured using an oxygen electrode after formate spiking, in presence of 1.29 to 27.1 µM HQNO (b) or 0.16 to 7.6 mM KCN (c). 100% corresponds to 153 nmol O₂/min/mg. **d.** **e.** Michaelis Menten plot of *BsForCE1*<sup>F128-D132D</sup> (d) and *BsForCE1*<sup>ΔHMP</sup> (e) coupling formate oxidation to menadione reduction *in vitro*. Data from n = 3 biological replicates are presented as mean values +/- SD.

**f.** Conservation of hydrophobic and positively charged residues at the respective F128 and R132 positions in the DUF1641 family. Sequences from Interpro IPR012440 (DUF1641) family were clustered and restricted to 644 representatives using MMseqs2 (Many-against-Many searching, https://toolkit.tuebingen.mpg.de/tools/mmseqs2) online software (minimum sequence identity>0.5; minimum alignment coverage>0.8). Then, multiple sequence alignment (MSA) was performed using Clustal Ω (default parameters). Finally, HMM logo was generated using http://skylign.org/ with the previously described MSA (from K<sup>118</sup> to Q<sup>155</sup>, *BsForE1* numbering) to consider both helix 7 and 8. Each stack represents one residue position within the MSA. Height of a letter within a stack represents the frequency of the corresponding residue at this position. The sequence of *BsForE1* is indicated below the HMM logo and helices 7 and 8 are indicated.
**g.** X-ray structure of *BsForCE1*<sup>ΔHMP</sup> superimposes with *BsForCE1*. *BsForCE1*<sup>ΔHMP</sup> is colored according to the b-factor value while *BsForCE1* is colored in gray. For clarity, one ForC subunit is shown on both structures.
<figure><img src="image_20.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_24.png" /></figure>

**Extended Data Fig. 3** | **For CE interaction with lipids.** a. Crystallographic and b. cryo-EM lipid composition inside the cavity formed by ForE tetramer. Structures are displayed in surface for the protein residues. Lipids, menaquinone and FeS clusters are depicted as sticks. ForC and ForE proteins surfaces are colored in dark blue and orange respectively. Non-protein carbon, oxygen, sulfur and iron atoms are colored in light blue, red, yellow and brown, respectively. c. Crystallographic and d. cryo-EM ForE central cavity access. ForC and ForE proteins surface are colored in dark blue and orange respectively. Glycerophospholipid carbon, oxygen, and phosphorus atoms are colored in light blue,

red and orange respectively. e. Hydrophobic (left) and electrostatic (center and right) surface calculations of the ForCE protein. Hydrophobic and hydrophilic residues are colored in light orange and light blue respectively. The cutaway shows the internal surface of ForE lined with hydrophobic residues (left). Positively charged amino acids are colored in blue, negatively charged amino acids in red, and neutral residues in white. The membrane was positioned at the level of the C-terminal end of the ForE tube capped by fatty acids (center). The right view shows the electrostatic potential of the HMP interacting with phospholipids.
<figure><img src="image_25.png" /><figcaption>**Extended Data Fig. 4** | Comparison between the Q-site in ForC (a) and HucS (b). **a.** α-helices 223–236 and 240–257 of the ForC V-shape motif are represented in cartoon colored in dark blue with the side chain (T100 and E237). Electron transfer distance between the menaquinone and the His-coordinated [4Fe–4S] cluster is indicated. α-helices of ForE are colored in yellow and brown and numbered. **b.** α-helix 298–311 from the HucS subunit is represented in cartoon</figcaption></figure>

colored in dark blue and light blue for the two symmetry related molecules, with the side chain of Y301 and Y229 and the backbone carbonyl of K212. Electron transfer distance between the menaquinone and the distal [3Fe–4S] cluster is indicated. α-helices of HucM are colored in yellow and brown and numbered. **a.** and **b.** Clusters are represented in balls and sticks. Oxygen, sulfur and iron atoms are colored in red, yellow and brown respectively.
<figure><img src="image_26.png" /><figcaption>b.</figcaption></figure>

<figure><img src="image_27.png" /></figure>

**Extended Data Fig. 5** | Comparison between ForC and canonical formate dehydrogenase. **a.** Structural superimposition of ForC1 with FdsA from the FDH of *R. capsulatus* (6TGA) colored according to the RMSD value. The specific ForC1 regions (that is the V-shape) are colored in grey. **b.** Comparison of the active site of BsForCE1 (left) to the one of *R. capsulatus* FDH (right). Protein atoms

and *bis*-pyranopterin guanosine dinucleotides (PGD) atoms are represented in sticks. Molybdenum and sulfur atoms are represented in balls. Carbon, oxygen, nitrogen, sulfur and molybdenum atoms are colored in gray, red, blue, yellow and turquoise respectively.
<figure><img src="image_28.png" /></figure>

**Extended Data Fig. 6** | EPR and redox analysis of [Fe-S] clusters in BsForCE1.

a. Representative EPR spectra of BsForCE1 S = 1/2 [Fe-S] clusters. Simulations (red traces) are superimposed on experimental spectra (black traces). The *g* values of [Fe-S] clusters are indicated with vertical dotted lines. Experimental conditions: Temperature, 15 K (1, 3), 30 K (2), 100 K (4); microwave power, 100 mW (1), 10 mW (2–4); field modulation amplitude, 1 mT; microwave frequency, 9.4812 GHz (1), 9.4791 GHz (2), 9.4801 GHz (3), 9.4809 GHz (4); redox potential (pH = 6.0), –288 mV (1), –280 mV (2), –391 mV (3), –395 mV (4). Simulation parameters are given in Extended Data Table 1. b. and c. EPR-monitored redox titration experiments of [Fe-S] clusters in BsForCE1 buffered at pH = 6 (b) and pH = 7.5 (c). EPR signal amplitudes of the Fe/S₁ [4Fe-4S] (g₁ = 1.825, green filled squares), the proximal [4Fe-4S] (g₁ = 2.044, blue filled triangles), Fe/S₂ (g₂ = 1.964, red filled circles), and the [2Fe-2S] cluster (g₂ = 2.018, pink filled triangles) were measured against the ambient redox potential. They were fitted to Nernst plots (solid lines) as described in the Methods, yielding estimated midpoint potential values as given in Fig. 2b. Experimental conditions: 15 K (Fe/S₁ and Fe/S₂) or 50 K (proximal [4Fe-4S], [2Fe-2S]), 100 mW (Fe/S₁), 10 mW

<figure><img src="image_29.png" /></figure>

(proximal [4Fe-4S] at pH = 7.5, Fe/S₂, [2Fe-2S]), 1 mW (proximal [4Fe-4S] at pH = 6.0), modulation amplitude, 1 mT (Fe/S₁ and proximal [4Fe-4S] at pH = 6.0, Fe/S₂), 0.5 mT (proximal [4Fe-4S] at pH = 7.5, [2Fe-2S]). d. Low field region of EPR spectra of dithionite-reduced BsForCE1 buffered at pH 6.0 and poised at –245 mV (top) and –390 mV (bottom). The signal with a maximum at g<sub>eff2</sub> = 5.19 and a shoulder at g<sub>eff1</sub> = 5.45 is ascribed to a [4Fe-4S]⁻ cluster with an S = 3/2 ground state (Supplementary Note 2 and Supplementary Figs. 6, 7). Experimental conditions: temperature, 8 K, microwave frequency, 9.480 GHz, microwave power, 100 mW, field modulation amplitude, 3 mT, 4 scans. The inset shows a potentiometric titration of the g<sub>eff</sub> = 5.45 signal. Experimental data were fitted to an n = 1 Nernst curve using an E<sub>m</sub> = –275 mV, with an estimated accuracy of ± 20 mV. The signals from the S = 3/2 [4Fe-4S] (inset) and from the S = 1/2 proximal cluster (panel b) titrate with the same redox potential value (within the experimental error, Fig. 2b). Based on this similarity, they are assigned to the same cluster, that is, the proximal one, which consequently exists in a mixture of S = 1/2 and S = 3/2 spin states, as reported for clusters coordinated exclusively by cysteines or by a mixed 3-Cys and 1-His<sup>50,51</sup>.
<table><thead><tr><th></th><th>ForCE<br>X-ray crystallographic<br>structure</th><th>ForCE<br>CryoEM reconstruction</th></tr></thead><tbody><tr><td>ForE<br>C-ter region</td><td>crystallographically<br>related ForE α7</td><td></td></tr><tr><td>HMP-α7</td><td>crystallographically<br>related ForE</td><td></td></tr><tr><td>ForE<br>N-ter region</td><td></td><td></td></tr></tbody></table>

**Extended Data Fig. 7 | Comparison of ForCE lipid composition in the crystal structure (left) and cryoEM reconstruction (right).** Main chain of the protein is represented in cartoon with the side chain in sticks. Lipids are represented in sticks. Carbon atoms are colored in dark blue for ForC and orange for ForE. Oxygen, nitrogen, phosphorus and sulfur, are colored in red, blue, orange and

yellow respectively. For the crystallographic structure, Fo-Fc difference Fourier electron density map (omit map) is displayed as green mesh and contoured at 3σ. For the cryoEM reconstruction, Coulomb potential density is displayed as a beige semi-transparent surface and contoured at 4σ for ForE C-ter region, glycerophospholipid 1 and 2 and contoured at 3.4σ for ForE N-ter region.
a

<figure><img src="image_30.png" /></figure>

FDH NDH-2 SQR Hases Other

b

<figure><img src="image_31.png" /></figure>

Archaea Bacteria

c

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

d

FDH_#1_Bacillus-subtilis_O05398/1-186
FDH_#1_Staphylococcus-aureus_A0DV5KEM7/1-157
NDH2_#2_Bacillus-subtilis_O34633/1-140
NDH2_#2_Blastoplasma-marina_A0258E2C7/1-250
SQR_#4_Bacteroides-up-AD058KBD4/1-227
SQR_#1_Naturibium-salinum_A0703YXM7/1-213
Hase_#7_Mycobacterium-smeagmatis_AQGUM5/1-189
Hase_#17_Aerobacillus-pallidus_A0A164ACCG0/1-175

1 MAKAIKRJQK----------EVTFEEDQRKDRERFEDAL-ADHKAELLETNHMEGHNMECR---VLPLERGELFGCDKVDLILVKKADT---------EETANTEKNELLFGLTGGLMDVKQLE----------102
1 MAFRISSKRIR----------LEKSEEQIKLESNEVTEAL-AANKDSILKABLKLTLDDAK---LLDAENGAIRCRQVINKFAVELNK----------DIYTCLLSNMA$MVFLGLNVSDLS----------103
1 MPETIJDQTNAS----------VSQSQDQLIQLLKPEVQSLTVL-VDQLPKLTLLVNLITLK----------SYDPQSVATDEVLK----------SDTVGATTELE----------DPFENLQALNVLDHLPQLEKAAE$MSDDLPNL----------78
1 MINTTQARQSG----------SLAERLNDPRTQOMLHR-LOHAEVLDNLAMAQD----------DLEQARATQLELLFKKATDPFENLQALNVLDHLPQLEKAAE$MSDDLPNL----------117
1 MAEKDLQKQIDDIINKLDAILECATAQKRSLEREDLME$DANI$KDAFRSTVEELERQGVEL---NWDDEKLFITFLKMKNIGKFTFVLDTFESLYDLMQDLGPVANEYIOMIRKMEHFQKGYFEFFAEITRALDTITHY$GEDVKLLADNII----------152
1 MSETTEAPESTD-LEAAIAENP$EAAATVERLDAVNELDVLAEGTALSDFMVRELSATGAT---LAESADGLATDETAA$LAF$AVCENC----------DDLRAALDTFELQSGT-DELAVAFVC$LAATAALDDFMVASLACTG----------133
1 MASNGHSACQNAIDELPOISPVQGIRRRLDDQVAALNSL-LOHADLAVLVKLCDCFVFRGGDIAN$NLETSA$ICELKALNAA----------DTPIPAALAKKVDLAGLAN$LATLSGLGVKATPALNAVLDSLTDQRG----------130
1 MCEACKGAVME----------TSIDHLLVEKLSDPRTMEQLILH-LOKLENVTFLEDMLEENFLQRGPEFADSINEEVVLLRQSISRPEVYTRF----------ENALTALHKMQEFLDSPVQQLELIKSD----------VLDVRS----------114

FDH_#1_Bacillus-subtilis_O05398/1-186
FDH_#1_Staphylococcus-aureus_A0DV5KEM7/1-157
NDH2_#2_Bacillus-subtilis_O34633/1-140
NDH2_#2_Blastoplasma-marina_A0258E2C7/1-250
SQR_#4_Bacteroides-up-AD058KBD4/1-227
SQR_#1_Naturibium-salinum_A0703YXM7/1-213
Hase_#7_Mycobacterium-smeagmatis_AQGUM5/1-189
Hase_#17_Aerobacillus-pallidus_A0A164ACCG0/1-175

103 - PILLKVNAGVASAVEQKNSE----------KKTGYFDIERSLK----------DPEINKSITLLPSFLKCMGQDTKELETTQPPHEQKHHQEPREKRGNNKRD----------186
104 - DFLNKVNGLCHVANDAS----------PKAKTTISLEGLVKL----------DDDMNRSIETVMLNMKLMGSRBI----------157
79 - PVKTAKEVATAITAEKDRRA----------EASNTGLFCLERMMLK----------DQPAQKLFRAFSNYLVLMNEKNENQK----------140
118 - JAVATOVDFWAELKADCVELEQSVRNGLRAALYLGGQIRREEDRLGYLLKSDVLSQH$VETVSLMC$ALSSCRQCSCHMPVPRMLCMGLL$ASR----------D$NTQRALAFAVQFGKCFNLCKDNHPSTSPDVKN----------250
133 - VTMDTKLNLTQPFMLRWNM----------AVNVFQGKLEDNIDNPEYSIWKAM-EMR$P$MKKCICFMITFLKNLSNENVAAIQG----------227
134 - AALGIEVQAATADDTRDCE----------TLTKVCGEAEREPERVGAGLCERV----------DPEVQYGLCYLLVASAIGHERADVERTAGGSQ----------213
133 - AEVLSALGDALVAARTSA----------PPAPRVGRCWMKTLAAAKDPDVGRGVSILLLVARVFQGV----------189
115 VQVIGKVSRSLYQASKEAAET----------CVRGICICRMTLS----------DPEVQPALLFNLFARNLSEISDA----------175

HMP/DUF1641

**Extended Data Fig. 8 | Analysis of the HMP/DUF1641 family.** a. The nodes/sequences are colored according to cluster type, b. kingdom of the organisms and c. sequence length. A good separation between archeal and bacterial sequences suggests a divergent evolution of the family. We also noted that NDH-2 organization seems to be present only in bacteria, and that some clusters tend to contain sequences of homogeneous length. d. Sequence alignment of

HMP/DUF1641-containing proteins. Two sequences from two different clusters from each type of functional arrangement, type I (clusters 1 and 3), type II (clusters 2 and 6), type III (clusters 4 and 6) and type IV (clusters 7 and 17), are aligned using Muscle and visualized in Jalview using the clustal coloring of residues and a transparency linked to conservation threshold of 30%. The position of the HMP/DUF1641 is boxed.
<figure><img src="image_33.png" /><figcaption>b.</figcaption></figure>

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

<figure><img src="image_35.png" /><figcaption>d.</figcaption></figure>

<figure><img src="image_36.png" /><figcaption>e.</figcaption></figure>

<figure><img src="image_37.png" /></figure>

**Extended Data Fig. 9** | Cryo-EM reconstruction. **a.** ForCE cryo-EM data processing workflow. **b.** Motion-corrected micrograph of ForCE complex. **c.** Fourier-shell-correlation plots for independently refined half-maps. Horizontal blue lines indicate Fourier-shell-correlation values of 0.143.

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

**d.** Examples of 2D classes of the final selection of particles. Number of the particles and estimated resolution in each 2D class are indicated. **e.** Guinier plot of the final homogeneous refinement indicating a sharpening B-factor of 119.5 Å². **f.** Angular distribution of the particles used for the final reconstruction.
Extended Data Table 1 | Comparison of the g-tensor characteristics of BsForCE1 [Fe-S] clusters with those of [Fe-S] clusters in other related enzymes

<table><caption>Comparison of the g-tensor characteristics of BsForCE1 [Fe-S] clusters with those of [Fe-S] clusters in other related enzymes</caption><thead><tr><th>Enzyme</th><th>Name of the cofactor (assignment, if any)</th><th>g values</th><th>Δg = g<sub>I</sub> - g<sub>II</sub></th><th>Reference</th></tr></thead><tbody><tr><td>BsForCE1</td><td>Fe/S<sub>1</sub></td><td>g<sub>1,2,3</sub> = 2.065, 1.889, 1.820</td><td>0.245</td><td>This work</td></tr><tr><td>MtFDH</td><td>Fe/S<sub>5</sub></td><td>g<sub>1,2,3</sub> = 2.09, 1.94, 1.84</td><td>0.25</td><td><sup>26</sup></td></tr><tr><td>CnFdsABG</td><td>A2 (C-ter [4Fe-4S])</td><td>g<sub>1,2,3</sub> = 2.095, 1.888, 1.843</td><td>0.252</td><td><sup>17</sup></td></tr><tr><td>Cpl[Fe-Fe] H2ase</td><td>FS4B (Central [4Fe-4S])</td><td>g<sub>1,2,3</sub> = 2.120, 1.909, 1.856</td><td>0.264</td><td><sup>27</sup></td></tr><tr><td>BsForCE1</td><td>Proximal [4Fe-4S]</td><td>g<sub>1,2,3</sub> = 2.044, 1.944, 1.893</td><td>0.151</td><td>This work</td></tr><tr><td>EcFDH-H</td><td>Proximal [4Fe-4S]</td><td>g<sub>1,2,3</sub> = 2.045, 1.957, 1.910</td><td>0.135</td><td><sup>28</sup></td></tr><tr><td>DaFDH</td><td>Fe/S<sub>1</sub> (Proximal [4Fe-4S])</td><td>g<sub>1,2,3</sub> = 2.046, 1.946, 1.882</td><td>0.164</td><td><sup>29</sup></td></tr><tr><td>CnFdsABG</td><td>A1 (Proximal [4Fe-4S])</td><td>g<sub>1,2,3</sub> = 2.045, 1.942, 1.897</td><td>0.148</td><td><sup>17</sup></td></tr><tr><td>RcFdsGBAD</td><td>A1 (Proximal [4Fe-4S])</td><td>g<sub>1,2,3</sub> = 2.046, 1.950, 1.892</td><td>0.154</td><td><sup>18</sup></td></tr><tr><td>DvHFdhAB</td><td>FeS<sub>1</sub></td><td>g<sub>1,2,3</sub> = 2.044, 1.937, 1.898</td><td>0.146</td><td><sup>30</sup></td></tr><tr><td>Complex I</td><td>N7 (eqvlt. Proximal [4Fe-4S])</td><td>g<sub>1,2,3</sub> = 2.045, 1.94, 1.89</td><td>0.155</td><td><sup>30,31</sup></td></tr><tr><td>BsForCE1</td><td>[2Fe-2S]</td><td>g<sub>II,II</sub> = 2.018, 1.937</td><td>0.081</td><td>This work</td></tr><tr><td>MtFDH</td><td>Fe/S<sub>3</sub></td><td>g<sub>1,2,3</sub> = 2.014, 1.94, 1.933</td><td>0.081</td><td><sup>26</sup></td></tr><tr><td>CnFdsABG</td><td>A5 ([2Fe-2S])</td><td>g<sub>1,2,3</sub> = 2.018, 1.948, 1.935</td><td>0.083</td><td><sup>17</sup></td></tr><tr><td>RcFdsGBAD</td><td>A5 ([2Fe-2S])</td><td>g<sub>1,2,3</sub> = 2.021, 1.943, 1.941</td><td>0.081</td><td><sup>18</sup></td></tr><tr><td>Complex I</td><td>N1b ([2Fe-2S])</td><td>g<sub>II,II</sub> = 2.02, 1.94</td><td>0.08</td><td><sup>31-34</sup></td></tr><tr><td>Ca[FeFe] H2ase</td><td>[2Fe-2S]</td><td>g<sub>II,II</sub> = 2.019, 1.936</td><td>0.083</td><td><sup>35</sup></td></tr></tbody></table>

Parameters used for the simulations of the EPR spectra shown in Extended Data Fig. 6a

<table><thead><tr><th>EPR spectrum</th><th>[Fe-S]</th><th>g values (g<sub>1,2,3</sub> or g<sub>II,II</sub>)</th><th>g-strain parameters FWHM<sub>1,2,3</sub></th><th>Proportion (%)</th></tr></thead><tbody><tr><td rowspan="2">1</td><td>Fe/S<sub>1</sub> [4Fe-4S]</td><td>2.065, 1.889, 1.820</td><td>0.050, 0.111, 0.070</td><td>86</td></tr><tr><td>Proximal [4Fe-4S]</td><td>2.045, 1.943, 1.894</td><td>0.009, 0.007, 0.010</td><td>14</td></tr><tr><td>2</td><td>Proximal [4Fe-4S]</td><td>2.044, 1.944, 1.893</td><td>0.017, 0.017, 0.019</td><td>100</td></tr><tr><td rowspan="4">3</td><td>Fe/S<sub>1</sub> [4Fe-4S]</td><td>2.060, 1.899, 1.827</td><td>0.077, 0.049, 0.140</td><td>36</td></tr><tr><td>Proximal [4Fe-4S]</td><td>2.044, 1.940, 1.896</td><td>0.011, 0.013, 0.012</td><td>21</td></tr><tr><td>Fe/S<sub>2</sub></td><td>1.998, 1.959, 1.894</td><td>0.028, 0.019, 0.034</td><td>23</td></tr><tr><td>[2Fe-2S]</td><td>2.018, 1.938</td><td>0.013, 0.009</td><td>20</td></tr><tr><td>4</td><td>[2Fe-2S]</td><td>2.018, 1.937</td><td>0.012, 0.009</td><td>100</td></tr></tbody></table>

Bs, Bacillus subtilis, Ec, Escherichia coli, Da, Desulfovibrio alaskensis, Cn, Cupriavidus necator, Mt, Methylosinus trichosporium OB3b, Ca, Clostridium acetobutylicum, DvH, Desulfovibrio vulgaris Hildenborough, Rc, Rhodobacter capsulatus, FDH, formate dehydrogenase, H2ase, hydrogenase. Parameters used for the simulations shown in Extended Data Fig. 6a. The numbering of the spectra in the first column is as shown in Extended Data Fig. 6a. Here are the references used in this table: refs. 52–60.
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No

Yes

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

Demonstrate how to render a vaccine ineffective

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /></figure>

Confer resistance to therapeutically useful antibiotics or antiviral agents

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

Enhance the virulence of a pathogen or render a nonpathogen virulent

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

Increase transmissibility of a pathogen

<figure><img src="image_64.png" /></figure>

<figure><img src="image_65.png" /></figure>

Alter the host range of a pathogen

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

Enable evasion of diagnostic/detection modalities

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

Enable the weaponization of a biological agent or toxin

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

Any other potentially harmful combination of experiments and agents
## Plants

**Seed stocks**

Report on the source of all seed stocks or other plant material used. If applicable, state the seed stock centre and catalogue number. If plant specimens were collected from the field, describe the collection location, date and sampling procedures.

**Novel plant genotypes**

Describe the methods by which all novel plant genotypes were produced. This includes those generated by transgenic approaches, gene editing, chemical/radiation-based mutagenesis and hybridization. For transgenic lines, describe the transformation method, the number of independent lines analyzed and the generation upon which experiments were performed. For gene-edited lines, describe the editor used, the endogenous sequence targeted for editing, the targeting guide RNA sequence (if applicable) and how the editor was applied.

**Authentication**

Describe any authentication procedures for each seed stock used or novel genotype generated. Describe any experiments used to assess the effect of a mutation and, where applicable, how potential secondary effects (e.g. second site T-DNA insertions, mosiacism, off-target gene editing) were examined.

## ChIP-seq

### Data deposition

<figure><img src="image_72.png" /></figure>

Confirm that both raw and final processed data have been deposited in a public database such as GEO.

<figure><img src="image_73.png" /></figure>

Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

**Data access links**

May remain private before publication.

For "Initial submission" or "Revised version" documents, provide reviewer access links. For your "Final submission" document, provide a link to the deposited data.

**Files in database submission**

Provide a list of all files available in the database submission.

**Genome browser session**

(e.g. UCSC)

Provide a link to an anonymized genome browser session for "Initial submission" and "Revised version" documents only, to enable peer review. Write "no longer applicable" for "Final submission" documents.

### Methodology

**Replicates**

Describe the experimental replicates, specifying number, type and replicate agreement.

**Sequencing depth**

Describe the sequencing depth for each experiment, providing the total number of reads, uniquely mapped reads, length of reads and whether they were paired- or single-end.

**Antibodies**

Describe the antibodies used for the ChIP-seq experiments; as applicable, provide supplier name, catalog number, clone name, and lot number.

**Peak calling parameters**

Specify the command line program and parameters used for read mapping and peak calling, including the ChIP, control and index files used.

**Data quality**

Describe the methods used to ensure data quality in full detail, including how many peaks are at FDR 5% and above 5-fold enrichment.

**Software**

Describe the software used to collect and analyze the ChIP-seq data. For custom code that has been deposited into a community repository, provide accession details.

## Flow Cytometry

### Plots

Confirm that:

<figure><img src="image_74.png" /></figure>

The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

<figure><img src="image_75.png" /></figure>

The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).

<figure><img src="image_76.png" /></figure>

All plots are contour plots with outliers or pseudocolor plots.

<figure><img src="image_77.png" /></figure>

A numerical value for number of cells or percentage (with statistics) is provided.

### Methodology

**Sample preparation**

Describe the sample preparation, detailing the biological source of the cells and any tissue processing steps used.

**Instrument**

Identify the instrument used for data collection, specifying make and model number.

**Software**

Describe the software used to collect and analyze the flow cytometry data. For custom code that has been deposited into a community repository, provide accession details.
**Cell population abundance**

Describe the abundance of the relevant cell populations within post-sort fractions, providing details on the purity of the samples and how it was determined.

**Gating strategy**

Describe the gating strategy used for all relevant experiments, specifying the preliminary FSC/SSC gates of the starting cell population, indicating where boundaries between "positive" and "negative" staining cell populations are defined.

<figure><img src="image_78.png" /></figure>

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

# Magnetic resonance imaging

## Experimental design

**Design type**

Indicate task or resting state; event-related or block design.

**Design specifications**

Specify the number of blocks, trials or experimental units per session and/or subject, and specify the length of each trial or block (if trials are blocked) and interval between trials.

**Behavioral performance measures**

State number and/or type of variables recorded (e.g. correct button press, response time) and what statistics were used to establish that the subjects were performing the task as expected (e.g. mean, range, and/or standard deviation across subjects).

## Acquisition

**Imaging type(s)**

Specify: functional, structural, diffusion, perfusion.

**Field strength**

Specify in Tesla

**Sequence & imaging parameters**

Specify the pulse sequence type (gradient echo, spin echo, etc.), imaging type (EPI, spiral, etc.), field of view, matrix size, slice thickness, orientation and TE/TR/flip angle.

**Area of acquisition**

State whether a whole brain scan was used OR define the area of acquisition, describing how the region was determined.

**Diffusion MRI**

<figure><img src="image_79.png" /></figure>

Used

<figure><img src="image_80.png" /></figure>

Not used

## Preprocessing

**Preprocessing software**

Provide detail on software version and revision number and on specific parameters (model/functions, brain extraction, segmentation, smoothing kernel size, etc.).

**Normalization**

If data were normalized/standardized, describe the approach(es): specify linear or non-linear and define image types used for transformation OR indicate that data were not normalized and explain rationale for lack of normalization.

**Normalization template**

Describe the template used for normalization/transformation, specifying subject space or group standardized space (e.g. original Talairach, MNI305, ICBM152) OR indicate that the data were not normalized.

**Noise and artifact removal**

Describe your procedure(s) for artifact and structured noise removal, specifying motion parameters, tissue signals and physiological signals (heart rate, respiration).

**Volume censoring**

Define your software and/or method and criteria for volume censoring, and state the extent of such censoring.

## Statistical modeling & inference

**Model type and settings**

Specify type (mass univariate, multivariate, RSA, predictive, etc.) and describe essential details of the model at the first and second levels (e.g. fixed, random or mixed effects; drift or auto-correlation).

**Effect(s) tested**

Define precise effect in terms of the task or stimulus conditions instead of psychological concepts and indicate whether ANOVA or factorial designs were used.

**Specify type of analysis:**

<figure><img src="image_81.png" /></figure>

Whole brain

<figure><img src="image_82.png" /></figure>

ROI-based

<figure><img src="image_83.png" /></figure>

Both

**Statistic type for inference**

Specify voxel-wise or cluster-wise and report all relevant parameters for cluster-wise methods.

(See Eklund et al. 2016)

**Correction**

Describe the type of correction and how it is obtained for multiple comparisons (e.g. FWE, FDR, permutation or Monte Carlo).
Models & analysis

n/a | Involved in the study

<figure><img src="image_84.png" /></figure>

Functional and/or effective connectivity

<figure><img src="image_85.png" /></figure>

Graph analysis

<figure><img src="image_86.png" /></figure>

Multivariate modeling or predictive analysis

Functional and/or effective connectivity

Report the measures of dependence used and the model details (e.g. Pearson correlation, partial correlation, mutual information).

Graph analysis

Report the dependent variable and connectivity measure, specifying weighted graph or binarized graph, subject- or group-level, and the global and/or node summaries used (e.g. clustering coefficient, efficiency, etc.).

Multivariate modeling and predictive analysis

Specify independent variables, features extraction and dimension reduction, model, training and evaluation metrics.