10: Biohydrogen - Hydrogenases

Introduction

In the last section, we described different ways to produce H₂ and the colors ascribed to them based on the environmental impacts. Many look to the production and use of H₂ to provide energy without releasing CO₂. H₂ can be used in fuel cells to power spacecraft and cars, as shown in the reaction below.

\begin{equation}
\begin{aligned}
& \mathrm{O}_2+4 \mathrm{H}^{+}+4 \mathrm{e}^{-} \longrightarrow 2 \mathrm{H}_2 \mathrm{O} \\
& \mathrm{H}_2 \longrightarrow 4 \mathrm{H}^{+}+4 \mathrm{e}^{-}
\end{aligned}
\end{equation}

Cars are already available that run on H₂ considered a zero-emission fuel. These fully electric cars use fuel cells powered by the oxidation of H₂ to produce electrical energy.

Figure \(\PageIndex{1}\):  https://afdc.energy.gov/vehicles/how...tric-cars-work

Given the scale needed, most H₂ is presently derived from the steam reformation of natural gas and the electrolysis of water. From a biochemical perspective, cells have evolved to make H₂ and use H₂ as an energy source. It's unlikely that direct microbial production of H₂ would meet society's energy needs. The 2023 International Energy Agency (IEA) report, "Hydrogen patents for a clean energy future", doesn't mention direct microbial production. However, much can be learned by studying how hydrogenases (H₂ases, Hyd), enzymes that make or use H₂, work. (Don't confuse hydrogenases with dehydrogenases that directly use NAD⁺/NADH and FAD/FADH₂) for redox reactions. Transition metal active site mimetics can be made as potential catalysts for more industrial-level production of H₂.

Although the reversible formation of H₂ involves the most elemental particles in chemistry, H⁺ and e^-, the biological reactions that produce and consume H₂ are complex. Before we proceed, let's see how these reactions are similar to other biochemical reactions and pathways we have already discussed.

Use of H₂ as a source of electrons for reduction reactions.  

Each hydrogen in H₂ has an oxidation number of 0. Each hydrogen can be oxidized to H⁺ (oxidation number +1) with the 2 electrons passed on to a substrate/cofactor or a sequential series of substrates with higher and higher standard reduction potentials (better oxidizing agents), leading to the formation of reduced products.  

H₂ + (substrate)_OX  →  2H⁺  +  (product)_RED

This general reaction is analogous to the mitochondrial electron transport chain, in which electrons are passed from a source (NADH) to oxidized forms of acceptors. The general reaction below shows each redox pair in the electron transport chain.

NADH/NAD⁺ →  FAD/FADH₂ →  UQ/UQH2  →  Cyto C_OX/Cyto C_RED  →   O₂/H₂O 

Some organisms have evolved to produce energy by the oxidation of H₂. This is analogous to photosynthetic organisms obtaining energy through the oxidation of water. In photosystem II,  oxygen in H₂O (oxidation number -2) gets oxidized by the oxygen-evolving complex to produce O₂ (oxidation number 0). Some redox pairs, starting with H₂O/O₂, are shown below for photosystem II.  

H₂O/O₂ →  P680/P680* →  (Plastoquione)_OX/(Plastoquione)_RED 

The first reaction is endergonic and requires as an energy source photons.

Use of H⁺ as a sink for electrons for oxidation reactions that produce H₂.  

H⁺ has an oxidation number of +1. Hence it can be reduced to H₂ (oxidation number of 0) as it gains electrons from substrates/cofactors, which get oxidized. This general reaction is shown below.  

2H⁺ + (substrate/cofactor)RED  →  H₂  + (substrate/cofactor)OX 

Many microorganisms can produce H₂ through variants of photosynthesis or through fermentation, both of which provide the two electrons needed. E. Coi has four hydrogenases (Hyd 1, 2, 3, and 4). It forms H₂ through two reactions catalyzed by:

• formate (HCO₂^-) dehydrogenase (FDH):  2HCO₂^- ⇌ 2CO₂ + 2H⁺ + 2e^- 
• hydrogenase (H₂ase):  2H⁺ + 2e^- → H₂ 

The C in formate has an oxidation number of +2 and is oxidized to CO_2, in which the C has an oxidation number of +4. 

The formate hydrogenlyase (FHL) complex contains both the formate dehydrogenase (FDH) and a hydrogenase (H₂ase) and reversibly interconverts HCO₂^– and H₂. The E. coli FHL-1 complex, which makes H₂ using fermentation, is shown below in Figure \(\PageIndex{2}\). The complex can be immobilized on a Macro-mesoporous inverse opal (IO) indium tin oxide (ITO) electrode (IO-IPO) or  ITO nanoparticles (NP), which can relay electrons.

Figure \(\PageIndex{2}\): Katarzyna P. Sokol et al. J. Am. Chem. Soc. 2019, 141, 44, 17498–17502.  https://doi.org/10.1021/jacs.9b09575.  CC-BY license

Panel (a) shows the biological E. coli FHL-1 complex. FdhF, [Mo]-FDH; B/F/G, Fe–S cluster-containing proteins; HycE, [NiFe]-H₂ase; HycD/C, membrane proteins. (17) 

Panel (b) shows a IO-ITO|FDH||IO-ITO|H₂ase cell: IO-ITO|FDH wired to IO-ITO|H₂ase electrode.

Panel (c) shows a FDH–ITO–H₂ase nanoparticle (NP) system with enzymes immobilized onto ITO NP in solution. Species size not drawn to scale.

All you need to synthesize H₂ are 2 protons and 2 electrons (potentially derived from photosynthesis). Let's take a deeper look at the hydrogenase that catalyzes H₂ production.

Hydrogenases (H₂ases)

Hydrogenases catalyze the reversible conversion of 2H⁺ →  H₂.   A hydrogenase database, HydDB, a web tool for hydrogenase classification and analysis of sequence, shows their high diversity and metabolic roles. There are three classes of hydrogenases, the Ni-Fe (most abundant, primarily for H₂ conversion to 2H⁺), the Fe-Fe (highest k_cat for H₂ production), and the single Fe hydrogenases, as shown in Table  \(\PageIndex{2}\) below. We won't discuss the single Fe hydrogenases.

Dan Søndergaard et al., Scientific Reports volume 6, Article number: 34212 (2016). Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

The three main types have different main functions in general. The Ni-Fe, Fe-Fe, and Fe H2ases generally oxidize H₂, produce H₂ and promote H^- (hydride) transfer, respectively, as shown in Figure \(\PageIndex{3}\) below. 

Figure \(\PageIndex{3}\):   The active site structures of [NiFe] H₂ases that mainly catalyze H₂ oxidation reactions, [FeFe] H₂ases that mainly catalyze H₂ evolution reactions, and [Fe] H₂ases that catalyze H− transfer to the substrate via heterolytic H2cleavage. X, possible H₂ active sites; Y, methenyltetrahydromethanopterin; GMP, guanosine monophosphate. Seiji Ogo et al., Science Advances.(2020).  DOI: 10.1126/sciadv.aaz81.  Creative Commons Attribution-NonCommercial License 4.0 (CC BY-NC).

Much effort has been devoted to making transition state analogs of the active site to act as catalysts for H₂ production for fuel cells. Transition metal catalysts that mimic the structures and activities of the three hydrogenases have been made. Three specific ones are shown below in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): The differing reactivity of the three isomers. Y′, methylene blue [MB]⁺.Seiji Ogo et al., ibid.

The ligand containing P and PH is bis(diphenylphosphino)ethane.

First, we will explore the Ni-Fe hydrogenases.

Ni-Fe H₂ases (Hyd): 

We'll discuss two examples of Ni-Fe H₂ases

Group 1a periplasmic (membrane-bound) hydrogenases - MBH

These are used in fuel cells and H₂-producing devices since they can adhere to surfaces that can be useful heterogeneous (not in solution) catalysts. Some also are damaged by O₂. The enzyme consists of a large subunit) found in the periplasm and small subunit, which anchors the protein in the plasma membrane of bacteria.  .  This enzyme oxidizes H₂:  H₂ → 2H⁺+2e⁻. The electrons enter the bacterial respiratory chain through quinones. The transmembrane part of the small subunit binds cytochrome b, which is involved in electron transfer with the quinones, as we saw in Complex II of mitochondrial electron transport. Some soil bacteria (like Ralstonia eutropha,) can use  H₂ as their sole energy source. The orientation of a NiFe MBH within a bacterial cell is shown in Figure \(\PageIndex{5}\) below.

Figure \(\PageIndex{5}\):  The orientation of a NiFe MBH within a bacterial cell. Lindsey A. Flanagan^* and Alison Parkin. Biochem Soc Trans. 2016 Feb 15; 44(1): 315–328 (2016). doi: 10.1042/BST20150201  Creative Commons Attribution Licence 3.0.

Panel (A) shows a cartoon depiction of how a NiFe MBH is located within the cytoplasmic membrane, with white boxes representing the redox active metal centers and blue, orange and purple blocks indicating the large, small and cytochrome subunits, respectively.

Panel (B) shows how the E. coli hydrogenase-1 large (blue ribbon), small (orange ribbon) and cytochrome (purple ribbon) subunits can interact. 

Figure \(\PageIndex{6}\) shows an interactive iCn3D modelof the O₂-Tolerant Membrane-Bound Hydrogenase 1 from Escherichia coli in Complex with Its Cognate Cytochrome b (4GD3). The same color coding is used for the subunits as in the above figures.

Figure \(\PageIndex{6}\): O₂-Tolerant Membrane-Bound Hydrogenase 1 from Escherichia coli in Complex with Its Cognate Cytochrome b (4GD3). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...9p4CSQRDPM8nL7

The large and small chains of hydrogenase are shown in blue and orange, respectively, while cytochrome b is sown in magenta. Cofactors and key metals are shown in spacefill. F4S is the Fe4-S3 cluster, SF4 is the iron-sulfur cluster, HEM is hemoglobin, FCO is carbonmonoxide-(dicyano) Fe, and Ni is nickel. 

The biological (functional) unit consists of two heterodimers. Under low O₂ levels, one dimer can reactivate the other if exposed to O₂. The enzyme is found in the highest concentration during anaerobic fermentation. Remember that E. Coli is a facultative anaerobe and can shift its metabolic pathways to fit conditions. Perhaps its primary role is to reduce O₂ to water and protect enzymes sensitive to it. The function of Cytochrome b may be mostly to anchor the dimeric H₂ase in the membrane.

A bifurcating Ni-Fe H₂ase

These enzymes are more complicated. They oxidize H₂ and move the two electrons through a complicated path that bifurcates electron flow to different substrates/cofactors. They move an electron to a low-potential (i.e. not a great oxidizing agent), high-energy species, which gets reduced in an endergonic process. The other electron simultaneously moves to a high-potential (i.e., great oxidizing agent), low-energy species that also gets reduced in an exergonic process. The overall electron transfer is thermodynamically favorable. An example might prove helpful. NADH (E^0' = -280 mV, higher potential) can reduce the protein ferredoxin (E^0' = -500 mV, lower potential), which can then pass its electrons in other reactions, including the formation of H₂, CH_4, and NH₃. ATP is not required.

One example of a bifurcating Ni-Fe H₂ase is the NiFe-HydABCSL protein from the bacteria A. mobile. The general structure of the pentameric form of the functional decamer is shown in Figure \(\PageIndex{8}\) below.

Figure \(\PageIndex{8}\): Structure of the A. mobile NiFe-HydABCSL pentamer. XIANG FENG et al. SCIENCE ADVANCES.  2022.   DOI: 10.1126/sciadv.abm7546.  Creative Commons Attribution License 4.0 (CC BY). https://creativecommons.org/licenses/by/4.0/

The five subunits are called HydA (Hyd = hydrogenase), HydB, HydC, HydL (large subunit), and HydS (small subunit). Pane (A) shows the domain structure of the five subunits. The NiFe-HydB NTD and CTDs are partially flexible, as indicated by dashed outlines. Panel (B) shows the subunit organization of the NiFe-Hyd complex and their associated cofactors.

NiFe-HydABCSL hydrogenase can reversibly oxidize H₂ with the two electrons reducing ferredoxin in an endergonic process and reducing NAD in an exergonic process. FMN is surrounded by an FeS cluster and appears to be the center of bifurcation.  The reaction is as follows:

• The HydL oxidizes H₂ with two electrons passing through the FeS centers in HydA to HybB.
• The electrons are passed to FMN, where the bound NAD gets reduced.  

Figure \(\PageIndex{9}\) jdkfjdkjfdkjff

Figure \(\PageIndex{9}\): Proposed mechanism of electron bifurcation/confurcation in A. mobile NiFe-HydABCSL.

(A) Overall electron transfer pathway, highlighting the three branches of the electron transfer path. The mid-potential path is a black dashed line, the exergonic path is a blue dashed line, and the endergonic path is a red dashed line. (B) Conformational changes in the HydBC bifurcation core from the electron bifurcation state (BR state) to the electron transduction state (PB state).

Figure \(\PageIndex{10}\) shows an interactive iCn3D modelof the electron bifurcating Ni-Fe hydrogenase complex HydABCSL in FMN/NAD(H) bound state 7T30

Figure \(\PageIndex{10}\): Electron bifurcating Ni-Fe hydrogenase complex HydABCSL in FMN-NAD(H) bound state (7T30). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...G9nKGwqvuuh6G7

Figure \(\PageIndex{11}\) shows an interactive iCn3D modelof the cofactors in the electron bifurcating Ni-Fe hydrogenase complex HydABCSL in FMN/NAD(H) bound state 7T30

Figure \(\PageIndex{11}\): Cofactors in the electron bifurcating Ni-Fe hydrogenase complex HydABCSL in FMN-NAD(H) bound state (7T30). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...hHuzaWUDYCMnAA.

Zoom into the Ni-Fe center catalytic site. The ligands that form coordinate covalent bonds to the Fe are called FCC, or carbon monoxide-(dicyano)-Fe  Figure \(\PageIndex{12}\) below. There are also bridging sulfurs between Fe and Ni.

Figure \(\PageIndex{12}\): Carbonmonoxo-dicyano-Fe and is shown in detail in 

Fe-Fe hydrogenases

These  enzymes catalyze a variety of reactions as illustrated in Figure \(\PageIndex{13}\) below.

Figure \(\PageIndex{13}\): [FeFe]-hydrogenases phylogeny and known functions.  Morra S. Front Microbiol. 2022 Mar 2;13:853626. doi: 10.3389/fmicb.2022.853626. PMID: 35308355; PMCID: PMC8924675.  Creative Commons Attribution License (CC BY)

A phylogenetic tree shows the phylogeny of [FeFe]-hydrogenase sequences from public databases, as previously proposed. Enzymes that have been experimentally characterized are indicated on the tree to show their relative position. The proposed physiological function of each enzyme is also presented, where known. Hyd, hydrogenase subunit; FdhF, formate dehydrogenase subunit; Fd_rex/ox, reduced/oxidized ferredoxin; NADH/NAD⁺, reduced/oxidized nicotinamide adenine dinucleotide.  They are found in prokaryotic and eukaryotic microorganisms, but not in Archaea. 

These are the most active for H₂ production with a k_cat around 10,000 s^-1. They contain a Fe₂S₂ cluster with CO and CN ligands forming bonds to the iron with the iron ions bridged by a -SCH₂-NH-CH₂S- (aza-dithiolate). A cysteine links the Fe2S2 to a Fe₄S₄ cluster. These two are called the H-cluster (or [Fe]_H. Within this class are a soluble, monomeric cytoplasmic form, a heterodimeric periplasmic form, and a soluble, monomeric chloroplastic form. This one has a ferredoxin, connecting it to the electron transport chain in photosynthesis. Some in this group using both NADH and ferredoxin are called bifurcating types, as they send two electrons from a donor in two different directions. More on this later.

They contain multiple FeS clusters. The H-cluster consists of a Fe₂S₂  linked to a Fe₄S₄ cluster (cubane-like) by a cysteine. The Fe₂S₂ group has CO and CN ligands, and the two Fe ions of Fe₂S₂ unit are coordinated by an azadithiolato ligand, as shown below in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\): Chemical structure of the H-cluster, which is the active site of the [FeFe] hydrogenase enzyme.  Rakesh C. Puthenkalathil et al., Phys. Chem. Chem. Phys., 2020, 22, 10447. https://pubs.rsc.org/fa/content/arti.../cp/c9cp06770a.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence

The Fe in the [2Fe2S] cluster is linked to the cubane [4Fe-4S] and has six ligands, so it is saturated. The other Fe has an extra coordination site denoted by X, which can bind H⁺ or H₂. The cluster is buried in a hydrophobic catalytic site which helps restrict O₂ access.

As we did for the Fe-Ni H₂ases, we will study two examples of Fe-Fe H₂ases.

Fe-Fe hydrogenase (CpI) from Clostridium pasteurianum

Figure \(\PageIndex{15}\) shows an interactive iCn3D modelof the H-Cluster (HC1) of Fe-Fe hydrogenase (CpI) from Clostridium pasteurianum (1FEH).

Figure \(\PageIndex{15}\): H-Cluster (HC1) of Fe-Fe hydrogenase (CpI) from Clostridium pasteurianum (1FEH). (Copyright; author via source). Click the image for a popup or use this external link:  https://structure.ncbi.nlm.nih.gov/i...TksW8FGn7crh39

In this model, the CN ligands are all displayed as CO. The sulfurs are shown in green. Hover over the atoms/ions to identify them.   (In iCn3D, choose, Select, Select on 3D, atom). The [4Fe-4S] subcluster forms coordinate covalent bonds with four cysteines (300, 355, 499, and 503) with one cysteine (503) forming a bridge to the [2Fe] cluster. The Fe ions in that cluster have an octahedral arrangement of ligands surrounding them. One of the ligands is water (no connecting C atom).  

Figure \(\PageIndex{16}\) shows an interactive iCn3D modelof the  Fe-Fe hydrogenase (CpI) from Clostridium pasteurianum (1FEH)

Figure \(\PageIndex{16}\):  Fe-Fe hydrogenase (CpI) from Clostridium pasteurianum (1FEH). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...QJsoZ8zbvkpDc7.

As we mentioned above, the net reaction is:

2H⁺ + (substrate/cofactor)RED  →  H₂  + (substrate/cofactor)OX 

Many microorganisms can produce H₂ through variants of photosynthesis or through fermentation, both of which provide the two electrons needed. E. Coi has four different hydrogenases, (Hyd 1, 2, 3 and 4). It forms H₂ through two reactions catalyzed by:

• formate (HCO₂^-) dehydrogenase:  2HCO₂^- ⇌ 2CO₂ + 2H⁺ + 2e^- 
• hydrogenase 3:  2H⁺ + 2e^- → H₂ 

Figure \(\PageIndex{17}\) below shows a reaction scheme for the production of H₂ linked to photosystem I in the chloroplast of microalgae under anaerobic conditions.  It starts with absorption of a photon by P700, which in the excited state transfers an electron to a 4Fe4S cluster in ferredoxin, which then passes an electron to the HC-cluster and then onto H⁺.

Figure \(\PageIndex{17}\):  Schematic representation of electron flow from Photosystem I to an [FeFe]-hydrogenase via a ferredoxin redox mediator (Photosystem I). JuanAmaro-Gahete et  al.,Coordination Chemistry Reviews. 448, December 2021. https://doi.org/10.1016/j.ccr.2021.214172.  Creative Commons CC-BY

A possible mechanism for the formation of H₂ in the H clusteris shown below in Figure \(\PageIndex{18}\) below.

Figure \(\PageIndex{18}\):  Proposed mechanistic cycle for hydrogen evolution in the H cluster by [FeFe]-hydrogenase adapted from Lubitz et al. JuanAmaro-Gahete et  al., ibid

Start at the top left which shows the resting oxidized state. In the enzyme's most oxidized resting system (H_ox), the [4Fe4S] cubane is in a 2 + oxidation state while the catalytic subcluster [2Fe] is a mixed-valence Fe^IFe^II state. The first one-electron reduction results in the formation of the H_red state, where the [4Fe4S] subcluster is reduced to a 1 + oxidation state. Protonation of the N of aza-propane-1,3-dithiolate ligand (adt-N) triggers an intramolecular charge shift to form H_redH⁺ in which the [4Fe4S] cubane is in the 2 + state and the [2Fe] subsite reduced to a homovalent Fe^IFe^I state. Subsequent one-electron reduction of the subcluster [4Fe4S] gives rise to the “super-reduced” state H_sredH⁺. In the next step of the catalytic cycle, an intermediate hydride state [H_hyd] is formed by an intramolecular proton shift from the adt-N to the distal iron F_d.  This process is coupled to an electron rearrangement in the [2Fe] subsite, leading to a formal Fe^IIFe^II oxidation state. Addition of a second proton coupled to another charge shift from the reduced [4Fe4S] to the [2Fe] subsite either in one or two discrete steps gives rise to [H_hydH⁺] that is characterized by a formal Fe^IFe^II oxidation state. At this point, there is an equilibrium between the H_hydH⁺ and H_ox[H₂] in which the hydride and the proton are combined into a hydrogen molecule at the distal iron of the system. The catalytic cycle is closed by H₂ release, returning to the initial H_ox configuration.

A bifurcating [Fe-Fe] hydrogenase from Thermotoga maritima (HydABC)

This enzyme, functionally a heterododecamer,  uses NADH as a source of electrons, which passes electrons to FMN, the bifurcation site, with an electron going to oxidized ferredoxin  (Fd_ox) and another to H⁺s for reduction to FD_RED and H₂. The enzyme consists of a dimer of a trimer of subunits HydA, HydB, and HydC, with dimer (HydABC)₂ interacting with another (HydABC)₂ to form a heterododecamer, with both halves acting independently. The two trimers (HydABC) in the dimer (HydABC)₂ are connected by a [4Fe–4S] cluster. A flexible loop in the B and A chain has a "closed" and "open" bridge conformation with a nearby Zn²⁺ important in the loop conformation.  

Figure \(\PageIndex{19}\) below shows the cryo-EM structure of the HydABC tetramer and the arrangement of the redox cofactors. 

Figure \(\PageIndex{19}\): Cryo-EM structure of the HydABC tetramer and arrangement of the redox cofactors. Chris Furlan et al. (2022)  eLife 11:e79361.  https://doi.org/10.7554/eLife.79361.  Creative Commons Attribution License

Panel (A) shows the unsharpened 2.3 Å map of Hyd(ABC)₄ with D2 symmetry enforced, showing a tetramer of HydABC heterotrimers. All four copies of HydB and C are colored blue and green, respectively. The four HydA copies that make up the core of the complex are orange, yellow, pink, and red. The top and bottom halves of the complex are constituted by dimers of HydABC protomers (each HydABC unit is a protomer); the two protomers within the same dimer are strongly interacting, while a weaker interaction is present between the top and bottom dimers.

Panel (B) shows the HydABC dimer highlighting the iron–sulfur clusters and flavin mononucleotide (FMN) constituting the electron transfer network.

Panel (C) shows the arrangement of redox cofactors within the protein complex, showing two independent identical redox networks (dashed circles); each redox network is composed of iron–sulfur clusters belonging to a Hyd(ABC)₂ unit consisting of two strongly interacting HydABC protomers.

Panel (D) shows a schematic of the electron transfer network of one of the two identical Hyd(ABC)₂ units showing edge-to-edge distances (in Å) between the various cofactors. Note that our structure is of apo-HydABC and contains only the [4Fe–4S]_H subcluster of the H-cluster. The 2H⁺/H₂ interconversion reaction in (B) illustrates the site at which this reaction occurs, but this will only occur in the fully assembled H-cluster, including [2Fe]_H.

Figure \(\PageIndex{20}\) shows an interactive iCn3D modelof the electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) (7P5H), using the same colors as the figure above.

Figure \(\PageIndex{20}\): electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) (7P5H). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...2hwCUnJ9BgwJi8.

Only the dimer (HydABC)₂ is shown. The A (gold), B (blue), and C (green) chains are colored as in the previous figure. The conformationally flexible loop at the C-terminal of a B chain in the closed state is shown in red. The gate also includes the C-terminal part of the A subunit near it.

Figure \(\PageIndex{21}\) shows the closed-bridge and open-bridge conformations of HydABC (the closed loop was shown in the model above).

Figure \(\PageIndex{21}\): closed-bridge and open-bridge conformations of HydABC from Thermotoga maritime.  

Panel (D) shows the HydB bridge domain in the open position and its fitted model. Panel (E) shows a Zn²⁺ hinge region and the two possible conformations of the HydB bridge domain, open (blue) and closed (light blue).

The similarities in cofactor arrangement in the Thermotoga maritime Hyd A, B and C subunits compared to the Nqo1, Nqo2, and Nquo3 subunits in Complex I from Thermus thermophilus (discussed in Chapter 19.1) are shown in Figure \(\PageIndex{22}\) below.

Figure \(\PageIndex{22}\): Comparison of the HydA, B and C subunits of the electron bifurcating [FeFe] hydrogenase from Thermotoga maritima with the Nqo3, 1 and 2 subunits from respiratory complex I from Thermus thermophilus.

Panel (A) shows the subunits HydA (red), HydB (blues), and HydC (green) overlaid with, respectively, Nqo3, Nqo1, and Nqo2 (all yellow) of complex I from T. thermophilus (Gutiérrez-Fernández et al., 2020, PDB: 6ZIY).

Panel (B) shows a comparison of the NADH-binding site of the Nqo1 subunit of complex I from T. thermophilus (light blue) with the flavin mononucleotide (FMN) site in HydB; the high similarity suggests NADH binds in the proximity of FMN in HydABC similar to complex I.

Panel (C) shows an electron transfer network in HydABC compared to complex I from T. thermophilus with edge-to-edge distances indicated in bold. The red, blue, and green dotted lines indicate the cofactors present in the HydA (Nqo3), HydB (Nqo1), and HydC (Nqo2) subunits, respectively. Note that our structure is of the apo-HydABC and lacks the [2Fe]_H subcluster of the H-cluster. The 2H⁺/H₂ interconversion reaction in (C) illustrates the site at which this reaction occurs, but this will only happen in the fully assembled H-cluster, including [2Fe]_H.

Here is a link to a video showing the conformational change observed between the ‘Bridge closed forward’ (7P8N) and ‘Open bridge’ (7PN2) classes.

In the video, the HydB C-terminal iron–sulfur cluster domain is colored blue, and the HydA C-terminal iron–sulfur cluster domain is colored orange. The zinc ion (gray sphere) and ligating residues (three cysteine ligands and one histidine) are also shown. The location of the HydA C-terminal domain when the bridge is open is unknown, so it is shown transparently in both states for reference.

The geometric separation of catalytic sites and the bifurcation mechanism prevents these thermodynamically favored reactions from happening

• H₂ production from ferredoxin oxidation (in the absence of NADH oxidation)

• NAD⁺ reduction by H₂ (in the absence of ferredoxin reduction)

• ferredoxin oxidation by NAD⁺

Oxygen Sensitivity of Fe-Fe H₂ases

We have alluded to the fact that Fe-Fe H₂ases can be sensitive to O₂.  A possible mechanism involves the interaction of one the Fe ions (Fe_d, the distal Fe) with oxygen, leading to the formation of damaging free radicals. As CO binds more strongly than O₂ to the iron in hemoglobin, its interaction with the H-center can help protect the H₂ases.  Sulfides can also afford protection.  These mechanisms are illustrated in Figure \(\PageIndex{23}\): 

Figure \(\PageIndex{23}\): Oxygen tolerance strategies in [FeFe]-hydrogenases. Morra S, ibid.

Schematic representation of the H-cluster in the oxidized active state Hox (centre). In the absence of any exogenous protectant, numerous [FeFe]-hydrogenases undergo irreversible inactivation due to H-cluster damage with loss of Fe atoms (red pathway); carbon monoxide acts as a protective agent due to its ability to form Hox-CO, by binding reversibly to the H-cluster at the same site as O₂ (purple pathway); in DdH, a similar mechanism occurs when sulphide binds to the H-cluster forming Hinact, via the Htrans intermediate (orange pathway); in CbA5H, a conformational change in the protein structure allows for a conserved cysteine to directly bind to the H-cluster, forming Hinact (green pathway). Fe_p, proximal iron atom; Fe_d, distal iron atom; Cys, cysteine residue.