Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

doi:10.21203/rs.3.rs-1116484/v1

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Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

https://doi.org/10.21203/rs.3.rs-1116484/v1

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Electron-bifurcation is a fundamental energy conservation mechanism in nature. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic primarily due to the lack of structural information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure is a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC heterotrimers electrically connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and proton reduction. Symmetry expansion identified two conformations of a flexible iron-sulfur cluster domain: a “closed bridge” and an “open bridge” conformation, where a Zn²⁺ site may act as a “hinge” allowing domain movement. Based on these structural revelations, we propose two new mechanisms of electron-bifurcation in HydABC.

Electron bifurcation splits a pair of electrons from a single two-electron donor to two different spatially separated electron acceptors with one being at a lower redox potential than the donor (1). This process drives thermodynamically unfavourable (endergonic) redox reactions by directly coupling them to energetically favourable (exergonic) redox reactions and, hence, it represents an alternative energy coupling mechanism to the well-known chemiosmotic coupling principle (2). Enzymes using electron-bifurcating are found in numerous biochemical pathways including respiration, photosynthesis, methanogenesis and the Wood-Ljundahl pathway of acetogensis (3–5). The process of electron bifurcation represents an exquisite example of how biochemical systems can use thermodynamic driving forces in a flexible and efficient manner and bifurcating enzymes hold potential as ‘molecular transformers’ in synthetic biology applications.

Electron bifurcation was first described in the Q-cycle of the respiratory complex III where the two electrons originating from the oxidation of ubiquinol are bifurcated via a high potential pathway to cytochrome c, and via a low potential pathway to reduce ubiquinone to ubiquinol (6, 7). This process has recently been discovered in a number of other enzymes where an exergonic electron transfer process is used to drive an endergonic one (3–5). Many of these enzymes have been proposed to utilize flavin-based electron bifurcation (FBEB), in which a flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) cofactor serves as the branch point for electrons. It first accepts a hydride from an intermediate potential redox couple (typically NAD(P)H) and then sends one electron down a high potential pathway, generating an unstable, low potential semi-reduced FMN, with low enough reducing power to send the second electron down a low potential pathway. The importance of FBEB in microbial metabolism and energy conservation is well acknowledged, but its mechanism is still poorly understood, with only a few examples so far being studied in detail, such as butyryl-CoA dehydrogenase-electron-transferring flavoprotein complex (Bcd-EtfAB) and Fd-dependent transhydrogenase (NfnI) (8).

The heterotrimeric [FeFe] hydrogenase, HydABC, from Thermotoga maritima is a soluble cytoplasmic enzyme involved in fermentation, which accepts electrons from the one-electron carrier ferredoxin (E_m = -453 mV at 80 °C, (9)), which is reduced during pyruvate metabolism, and the two-electron carrier NADH (E_m = -320 mV at pH 7, (9)), produced during glucose metabolism, to reduce protons to hydrogen (Eº’ = -420 mV at pH 7, (9)). The mechanism by which this enzyme functions is debated, however, the predominant view is that an FBEB mechanism is operative (10). The biochemically characterized FMN at the NADH binding site is unlikely to act as a bifurcating center for the following reasoning. As mentioned above a bifurcation centre should accept two electrons from at intermediate potential and donate one at high potential and the second at low potential. Since the FMN accepts electrons from H₂ (intermediate potential) and directly donates a hydride to NAD⁺ (high potential), there is no way for the FMN to donate a second electron to ferredoxin.

Therefore, the site of bifurcation has been speculated to be a second flavin cofactor (11). However, biochemical studies do not corroborate the presence of the second flavin (12). In another hypothesis, the hydrogen conversion center, the so-called H-cluster, which also undergoes two-electron redox chemistry, is speculated to be the electron-bifurcation center (7). However, spectroscopic studies suggest that the H-cluster of HydABC has similar redox properties to the non-bifurcating [FeFe] hydrogenases, having a stable one-electron reduced state, and is, therefore, also unlikely to be the site of bifurcation (12).

As structural data would reveal the complex arrangement of redox cofactors in this enzyme and provide a stronger basis for understanding the mechanism of electron bifurcation, here we report a 2.3 Å resolution structure of HydABC based on electron cryo-microscopy (cryo-EM) of single particles. The cryo-EM structure suggests a synergic coupling between two HydABC heterotrimers connected through the His-ligated [4Fe-4S] cluster in the HydA subunit, which may allow functionally important electron exchange between the two heterotrimers. The structure also reveals flexible C-terminal (CT) domains in HydA and HydB (here named “bridge” domains), which contain additional iron-sulfur clusters. These domains interact through non-covalent interactions and may provide a second electron transfer pathway. Thus, this structure provides details of the arrangement of the redox clusters in HydABC, based on which two novel mechanisms of electron-bifurcation are proposed.

The structure of HydABC

The heterologous production of apo-HydABC in Escherichia coli was described recently (12). Here, we have used this heterologously expressed HydABC to prepare the cryo-EM grids. Following the grid imaging, data collection, and processing, we obtained a 2.3 Å resolution map when D2 symmetry was enforced (fig. S1, fig. S2, A). Into this, an atomic model of HydABC was constructed, starting with a homology model based on homologous subunits in bacterial complex I (12, 13), together with ab initio model building in regions of the highest resolution. Initially, the last 91 and 61 C-terminal (CT) residues of HydA and HydB, respectively, could not be built as they were not present in the homology model and had a low resolution in the map, indicating regions of high heterogeneity (explored later).

The processed cryo-EM map shows that the heterotrimeric HydABC forms a tetrameric complex, Hyd(ABC)₄ of HydABC heterotrimer units (protomers). Oligomerization of HydABC occurs through interactions between four HydA subunits in the core of the complex (Fig. 1, A see also Movie S1). Each HydA has extensive interactions with one adjacent HydA chain (buried surface area of 2,280 Å²), and minor interactions with another HydA chain (780 Å²) (Fig. 1, A). HydB is tightly bound to a single HydA (buried surface area of 1,232 Å², table S1) but with minor interactions between HydB of one heterotrimer and HydA and HydB in another heterotrimer. HydB and HydC extend outward from the core and form the four lobes clearly visible in the 2D class averages (fig. S3). The HydA core is the best resolved part of the map, consistent with the core being rigid and homogenous (fig. S4).

Based on the density map, each HydABC protomer appears to contain nine redox cofactors including five [4Fe-4S] clusters (one of which forms the [4Fe-4S] subcluster of the H-cluster), three [2Fe-2S] clusters, and one FMN. However, based on published Fe quantitation as well as published sequence analysis predictions we expect a total of seven [4Fe-4S] clusters (including the subcluster of the H-cluster) and four [2Fe-2S] clusters in each HydABC protomer (14, 15). According to sequence predictions, these missing clusters should be located in the less well resolved CT regions of the HydA and HydB subunits (discussed below) (14). Interestingly, a high-density site, likely a monometallic center, is found in the resolvable part of the HydB-CT domain. Inductively-coupled plasma mass spectrometry on the separately produced and purified HydB subunit identified 0.99 ± 0.43 Zn/protein and ≈14.2 ± 1.5 Fe/protein. As the observed Fe-content matches with the estimated Fe-content of HydB, which is expected to contain three [4Fe-4S] clusters and one [2Fe-2S] cluster (14 Fe/protein), these results allow us to assign the metal center as zinc (Zn²⁺). This is further supported by the identities of the ligating residues: three cysteines and one histidine in a tetrahedral coordination geometry (fig. S5, C) (16).

Cofactor arrangement in HydABC

Electron transfer chains, often connecting distant active sites, are composed of redox-active cofactors usually less than 14 Å apart to allow sufficiently fast electron tunnelling through the protein dielectric to sustain catalysis (17). In each HydABC heterotrimer, the spatially distant H-clusters and FMN centers are electrically connected via a chain of four FeS clusters (A1, A2, A3, and B2, see Fig. 1, D for cluster nomenclature). The edge-to-edge distances between all these clusters are < 15 Å and within a distance for electron transfer at physiologically relevant rates (Fig. 1, D). Among the three remaining FeS clusters, the [4Fe-4S] cluster from HydA (A4) lies at the interface of the two tightly interacting HydA chains, and the two [2Fe-2S] clusters from HydC (C1) and HydB (B1) subunits lie in the vicinity, but on the opposite side, of the FMN toward a dangling helix at the N-terminus of HydB (fig. S6). This helix has locally lower resolution indicating some structural heterogeneity. Similar structural features have been suggested to function as a “fishing rod” for catching ferredoxin in a “fly casting” mechanism in cyanobacterial complex I (18) and ferredoxin NADP⁺ reductase (19, 20). Furthermore, a formate dehydrogenase enzyme that is closely related to HydABC but which does not bind ferredoxin, is specifically missing this helix (21). Thus, we suggest that the N-terminus of HydB may be the region where ferredoxin binds and transfers electrons to the B1 cluster. Electrons from ferredoxin would then be transferred from B1 to C1, and possibly to the FMN. However, the distance between C1 and FMN is ~16 Å, which is at the theoretical limit for fast electron transfer (17).

Within the Hyd(ABC)₄ complex, there appear to be two redox networks, each composed of two electrically connected HydABC protomers, separated by at least 50 Å and held together by extensive HydA-HydA interactions (Fig. 1, B and C). The large distance between each network indicates there is no possibility for electrons to be exchanged and that they probably function independently (Fig. 1, C). The two tightly interacting HydABC protomers within the Hyd(ABC)₂ unit are electrically connected through the His-ligated [4Fe-4S] cluster (A4) in HydA (Fig. 1, B), part of the so-called Y-junction of iron-sulfur clusters (22). This junction is well conserved in a wide number of structurally related enzymes, but its function is unknown. In HydABC it is clear that the Y-junction connects the NADH and ferredoxin oxidation sites to the hydrogenase active site and to the neighboring protomer. The two A4 clusters are separated by 9.0 Å and have the possibility to allow overflow of electrons from one protomer to the other. An electrical connection between two identical protomers has already been observed in cytochrome bc₁ (23), called an electronic ‘bus-bar’, which is speculated to have a number of possible roles such as allowing the physiological function of the protein even after operational damage of one of the two protomers.

Structural comparison of HydABC with homologous proteins

The spatial arrangement of subunits HydA, B and C in the HydABC protomer is similar to that of subunits Nqo3, Nqo1, and Nqo2, respectively, in the NADH-oxidation (N) module of Thermus thermophilus (Tt) respiratory complex I (fig. S7). This comparison is useful because complex I is structurally well-characterised, but does not oxidize ferredoxin or carry out electron-bifurcation. Therefore, structural differences between the subunits of complex I an HydABC may reveal important insight into the mechanism of electron transfer in the latter. The individual subunits are structurally highly similiar, with the highest similarity between HydB and Nqo1 (rmsd 1.040 Å) (24), followed by HydC and Nqo2 (rmsd 1.152 Å) and the lowest similarity between HydA and Nqo3 (rmsd 1.294 Å) (Fig. 2, A). The remarkable structural similarities between HydB and Nqo1 subunits agree with their common evolutionary origins (25), and suggest that NADH oxidation follows a similar mechanism in both enzymes (Fig. 2, B). The structural differences between Nqo3 and HydA likely reflect the fact that the latter accommodates the hydrogenase H-cluster and facilitates oligomerization of the Hyd(ABC)₄ complex.

The structural similarities between HydABC and Tt respiratory complex I are also reflected by the FeS clusters positioning that is in excellent agreement in these two proteins (Fig. 2, C). However, in contrast to the Tt complex I, the HydABC protomers contain five additional FeS clusters. One of these additional clusters is a [4Fe-4S] cluster (A3) that electrically connects the [4Fe-4S] subcluster of the H-cluster (analogous to the cluster N7 in Tt complex I) with the rest of the electron transfer network. Another additional cluster is a [2Fe-2S] cofactor in HydB (B1) that is electrically connected to the [2Fe-2S] cluster in HydC (C1, analogous to N1a in Tt complex I); due to this connection and the proximity of HydC to the “bridge” domains (discussed later) it is likely that the [2Fe-2S] cluster in HydC has an important role in the mechanism of electron bifurcation, this is in contrast to its analogous N1a cluster in complex I, the role of which is unclear but is certainly not part of the main catalytic electron transport pathway (26, 27).

The HydA subunit has close structural homology (35% sequence identity) to the well-characterized monomeric non-bifurcating [FeFe] hydrogenase from Clostridium pasteurianum, CpI. In contrast to electron bifurcating [FeFe] hydrogenases, non-bifurcating [FeFe] hydrogenases use a single redox partner, typically ferredoxin. Aligning the two enzymes shows high similarity (rmsd 1.119 Å) and excellent conservation of the FeS clusters, including the A4 cluster, which connects neighboring HydA subunits in HydABC (Fig. 3). However, in CpI, for which ferredoxin is the only redox partner, the cluster homologous to A4 is thought to lead to the ferredoxin binding site (28). The multimerization of HydA blocks this site, so the two enzymes must have different ferredoxin binding sites. This re-arrangement is an example of how closely related systems may have different electron-transfer pathways formed by different multimerization of their subunits.

A bridging domain formed by the flexible C-termini of the HydA and HydB subunits

The core of the tetrameric HydABC complex is very well resolved, reaching a local resolution of 2.2 Å. However, the lobes formed from HydA and HydB subunits have substantially lower local resolution (~3 Å), due to increased heterogeneity (fig. S4) and low intensity, blurred map density was observed between the lobes of electrically connected HydABC protomers (fig. S8, A). To investigate the blurred regions, symmetry expansion followed by classification was explored to separate the different conformations into classes. Initial attempts to use D2 symmetry, to match the core, resulted in maps no better than before, however, using C2 symmetry revealed two classes with bridging density between the HydB lobes (Fig. 4, A) with local resolution similar to the lobes formed from HydA and HydB (Fig. 4, B). This bridging density breaks the rotational symmetry between the protomers in the Hyd(ABC)₂ unit, explaining why D2 symmetry expansion was ineffective. The two classes correspond to the bridge domain being formed between different HydB lobes: when rotated by 180°, the bridges are identical (Fig. 4, A and C). Despite extensive attempts, we were unable to find a class with both bridges in the closed conformation. The observation that both bridges cannot close simultaneously suggests that these behave as reciprocating elements. A similar observation was made for the Rieske domains in the bifurcating bc₁ complex (30).

To further explore the particles without a bridge a further classification was used (Fig. S8, B). It was possible to obtain a low-resolution map of a class where the HydB CT domain was found in an ‘open’ conformation (Fig. 4, D). The movement of the HydB C-terminal domain between the bridge-open and bridge-closed classes is shown in Fig. 4, E and Movie S2.

In the bridge-containing structure, the two C-terminal [4Fe-4S] clusters (named B3 and B4, Fig. 4, F) of HydB are close enough to exchange electrons with each other but are too far from the next nearest FeS clusters, such as cluster C1 (≈ 35 Å away) or cluster A5 (≈ 32 Å away). Furthermore, cluster A5 is completely electrically isolated with all the nearest clusters being >30 Å away. Thus, unless the HydA and HydB bridge domains undergo substantial conformational changes, the FeS clusters A5, B3 and B4 cannot participate in electronic exchange with the rest of the enzyme.

The bridge structure is particularly interesting as it appears that the C-terminal cysteine residues of HydB responsible for coordinating [4Fe-4S] clusters in the bridge are conserved in all biochemically characterized electron-bifurcating [FeFe] hydrogenases (31, 32). However, they all lack the analogous part of the bridge domain in HydA, which contains the A5 cluster.

HydABC is not a typical flavin based electron bifurcating enzymesas it lacks a flavin center capable of accepting two electrons from an intermediate potential redox couple i.e. 2H⁺/H₂ (-410 mV at pH 7) and splitting them so that one electron goes toward a high potential couple i.e. NAD ⁺/NADH (-320 mV at pH 7), and the other goes toward a low potential couple i.e. ferredoxin (-450 mV). The hypothesis that a second flavin site i responsible for bifurcation (11) is neither supported by the biochemical experiments (12), nor by the cryoEM structure of HydABC presented herein: a single flavin (the FMN in HydB) that accepts a hydride from NADH, exists in this enzyme. The second hypothesis, that the H-cluster is the bifurcation center (7), is not supported by the fact that the H-cluster of HydABC shows similar behaviour to the H-cluster from non-bifurcating [FeFe] hydrogenases (12). Structural comparison of the HydA subunit (of HydABC) with the non-bifurcating [FeFe] hydrogenase CpI also reveals that the primary and secondary coordination spheres of the H-cluster are highly conserved in the two enzymes, thereby, supporting our previous conclusion (12).

Having excluded the H-cluster and FMN, we propose that the bifurcation site is an iron-sulfur cluster or a group of iron-sulfur clusters and are now able to provide two potential mechanisms, both involving the HydB-CT domain. This domain, carrying the B3 and B4 clusters, is found in all characterized electron-bifurcating [FeFe] hydrogenases but is absent in non-bifurcating NAD⁺-dependent multimeric [FeFe] hydrogenases (31, 32). Therefore, these clusters are considered an essential component of the mechanism. In Mechanism 1 (Fig. 5A), H₂ is oxidized by the H-cluster and electrons are sent down the core electron transfer pathway to the FMN. Meanwhile electrons in the adjacent protomer are picked up by cluster A´5, through a large scale conformational change and transferred to the B3/B4 clusters in the bridge. Another conformational change moves the B3/B4 clusters close to the B1/C1 clusters for electron transfer to ferredoxin while NAD⁺ is reduced to NADH at the FMN. In Mechanism 2 (Fig. 5B), electrons from H₂ oxidation at the H-cluster travel via the core electron transfer pathway to FMN/C1 and then to the B3/B4 clusters in the bridge causing the bridge to close. This then allows a second H₂ oxidation to reduce the FMN. NAD⁺ reduction triggers the bridge to reopen moving the reduced B3/B4 clusters close to the B1/C1 pair allowing electron transfer to ferredoxin.

In summary, our cryo-EM structure reveals essential information on the arrangement of cofactors and active sites within T. maritima HydABC, including inter-protomer electronic wiring. Using symmetry expansion, we have also observed two conformations of the HydB-CT domain, a domain that is unique to and conserved in bifurcating hydrogenases, consistent with mechanistically relevant conformational changes. These structural revelations open up new avenues for mechanistic views on electron-bifurcation wherein sophisticated arrangements of FeS clusters could act as the bifurcation-center. Such mechanisms may also be operative in other enzymes homologous to HydABC. By resolving these crucial structural details, the mechanism of bifurcation can now be tackled using site directed mutagenesis coupled to kinetic and spectroscopic studies.

Protein expression and purification

HydABC and HydB were produced heterologously in Escherichia coli BL21(DE3) ΔiscR cells under anaerobic growth conditions and purified in an anaerobic glovebox (Coy, 2% H₂ in N₂) using Streptactin (IBA) affinity chromatography and size exclusion chromatography (GE Healthcare) as previously described (12). For these studies, we did not incorporate the [2Fe]_H subcluster to form the holo-enzyme. Previous studies with the CpI [FeFe] hydrogenase showed essentially no difference in the protein structure between the apo-hydrogenase lacking the [2Fe]_H subcluster (29) Sample purity and quality were checked by SDS-PAGE and UV-vis spectrophotometry. Samples in 10 mM Tris-HCl, 150 mM NaCl, pH 8 were frozen at -80°C until further use.

Inductively-coupled plasma mass spectrometry (ICP-MS)

For ICP-MS, a sample of the HydB subunit, buffer exchanged into 10 mM MOPS pH 7 and concentrated to 621 µM, and a sample of 10 mM MOPS pH 7 were measured by Mikroanalytisches Laboratorium Kolbe (www.mikro-lab.de). The samples were digested using a CEM Model MARS6 microwave digestion unit and measured on an Agilent Model 7900 ICP-MS.

Grid preparation and imaging

1.2/1.3 UltrAuFoil grids were glow discharged (PELCO easiGlow) for 90 s on each side using atmospheric gas before mounting in Vitrobot (model IV) tweezers (Thermo Fisher Scientific). Individual HydABC aliquots were defrosted and 2.5 µL immediately placed onto the grid, blotted, and plunged into liquid ethane. 12 grids were prepared, varying blot time from 2 - 4 s with 0.75-1.5 mg mL⁻¹ protein; blot force was constant at -5. Following screening, a grid at 1 mg mL⁻¹ protein concentration was selected for data collection on a Titan Krios microscope operated at 300 kV with a K2 detector and energy filter. The energy filter was set to a 20 eV window. 3 exposures were collected per hole, and the autofocus routine was run every 10 µm. AutoCTF was used to correct for astigmatism and coma. 4790 movies of 48 frames each were collected. The total fluence was 57 electrons / Å².

Image processing

The Relion pipeline was used for all image processing. Whole micrograph motion correction and damage weighting was performed using the implementation of MotionCor2 in Relion (33). Initial CTF values were determined with CTFFIND4 (34) and particles were picked using a low resolution (≈10 Å) preliminary dataset that was previously collected (not described here). The early stages of 2D and 3D classification used images with the original pixel size downsampled from 0.85 Å to 3.4 Å/pixel. The particles were cleaned using two rounds of reference-free 2D classification. An initial model was generated in Relion and coarse 3D classification (7.5° sampling) without symmetry being enforced was used to remove broken particles. The particles were re-extracted with the original pixel size of 0.85 Å/pixel and 3D auto-refinement of these particles resulted in a 2.5 Å resolution structure when D2 symmetry was applied and after iterative CTF refinement of anisotropic magnification, trefoil and 4th order aberrations; astigmatism and defocus were fitted on a per-particle basis. The map displayed the features expected at such a resolution, with rotamers of many side chains being clear and water molecules being visible in well-resolved regions. The Bayesian polishing function of Relion-3.1 (35) resulted in a 2.3 Å resolution structure when D2 symmetry was applied (fig. S1); further CTF refinement led to no further gains in resolution. The final calibrated pixel size was 0.824 Å.

The HydA and HydB CT domains forming the “bridge” between lobes were found using the symmetry expansion tools in Relion. The high-resolution D2 refinement was used as a starting point. The relion_symmetry_expand command was used to apply a C2 symmetry operator to the particles in the refined .star file. A 20 Å low-pass filtered mask, generated from fitted atomic coordinates and expanded by 20 pixels with 6 pixels soft-edge, was then applied to half of the complex containing two tightly connected HydABC protomers with a complete electron transfer network. A mask was then created that included exclusively the two “bridges” densities in the Hyd(ABC)₂ unit (20 Å low-pass filter, 6 pixels soft edge), allowing a better 3D classification without losing any signal in the “bridges”. The resulting “bridged” classes (bridge backward and forward) were refined with C1 symmetry applying a 6 pixel soft edge mask that included the Hyd(ABC)₂ unit with two bridges, reaching a resolution of 2.8 Å for both the classes.

To explore the location of the HydB in the non-bridged class, a mask was created around the suspected area and used for classification and refinement (fig. S8, B). The improved map allowed an improved mask to be created for a final round of classification and refinement. The resulting map density is of insufficient quality for ab initio model building, but the strong FeS signals allowed the HydB CT-domain to be docked in place (Fig. 4, D).

Model building and validation

We used a homology model generated based on bacterial complex I (13) discussed in our recent paper on HydABC (12) as a starting point for model building. Here the Nqo3 subunit of complex I is related to HydA, Nqo1 to HydB, and Nqo2 to HydC. The map density was sufficiently strong to allow ab initio building of the non-conserved regions of HydA and HydB in the well-resolved parts of the D2 map, however without further classification many parts of HydB and HydC were poorly resolved. Model refinement was performed using Phenix real-space refinement. Phenix now automatically recognises the ligation between FeS clusters and cysteines, so it is no longer necessary to manually define these restraints or to provide the correct definition of the FeS geometry (36).

The “bridge” is formed from 91 residues of the CT of HydA and 61 residues of the CT of HydB. The HydA CT “bridge” domain has homology with the CT of HndA from the NADP-reducing hydrogenase complex in Desulfovibrio fructosovorans (37) and 82 CT residues of T. maritima HydC. The HydB CT “bridge” domain has homology with bacterial 2x[4Fe-4S] ferredoxin domains. In both cases, Phyre2 was used to build a homology model from this information, which was further built into the density, combined with the model for the rest of the complex built from the D2 map and refined (38).

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Acknowledgments

This work benefited from access to the Astbury Biostructure Laboratory, an Instruct-ERIC centre. Financial support was provided by Instruct-ERIC (PID 11666). We are grateful to Svetomir Tzokov, Charlotte Scarff, and Rebecca Thompson for assistance with data collection and Nigel Moriarty for assistance with FeS ligation during model building. We used computational resources provided by the Viking Cluster at the University of York and are grateful to the Research Computing team and Huw Jenkins for assistance with computing. We thank Mikroanalytisches Laboratorium Kolbe for ICP-MS measurements. We are grateful to Hannah Bridges, Laure Decamps and Patrícia Rodríguez Maciá for critical evaluation of the manuscript. J.A.B. and N.C. acknowledge funding from the DFG SPP 1927 “Iron−Sulfur for Life” project (Project No. BI 2198/1-1). The work was supported by the Max Planck Society (J.A.B., N.C. and W.L.), and in part a UKRI Future Leader Fellowship (JNB; MR/T040742/1) and JSPS KAKENHI (Grant number 16K21748 (H.O.).

Author contributions

Conceptualization: NC, HO, WL, JNB, JAB

Methodology: CF, NC, PG, JNB, JAB

Investigation: CF, NC, PG, JNB, JAB

Visualization: CF, PG, JNB

Funding acquisition: JNB, JAB

Project administration: JAB, JNB

Supervision: JAB, JNB

Writing – original draft: CF, NC, JNB, JAB

Writing – review & editing: CF, NC, PG, HO, WL, JNB, JAB

Competing interests

Authors declare that they have no competing interests.

Data and materials availability

Protein databank (PDB) files for the four model presented in this manuscript are available at https://www.rcsb.org/ under PDB ID 7P5H (D2 tetramer, 7P8N (Bridge closed forward), 7P91 (Bridge closed reverse), and 7P92 (Open bridge). Cryo-EM maps are available at https://www.ebi.ac.uk/pdbe/emdb/. All other data are available in the main text or the supplementary materials.

There is NO Competing Interest.

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Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

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Abstract

Figures

Introduction

Results

The structure of HydABC

Cofactor arrangement in HydABC

Structural comparison of HydABC with homologous proteins

A bridging domain formed by the flexible C-termini of the HydA and HydB subunits

Discussion

Online Methods

Protein expression and purification

Inductively-coupled plasma mass spectrometry (ICP-MS)

Grid preparation and imaging

Image processing

Model building and validation

References

Declarations

Additional Declarations

Supplementary Files

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