Structural basis for bacterial energy extraction from atmospheric hydrogen.

Diverse aerobic bacteria use atmospheric H2 as an energy source for growth and survival1. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments2,3. Atmospheric H2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily4,5. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H2 amid ambient levels of the catalytic poison O2 and how the derived electrons are transferred to the respiratory chain1. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H2 at the expense of O2, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H2 in ambient air.

Second and Outer Coordination Sphere Effects in Nitrogenase, Hydrogenase, Formate Dehydrogenase, and CO Dehydrogenase.

Gases like H2, N2, CO2, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N2, CO2, and CO and the production of H2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N2 fixation by nitrogenase and H2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere.

Oxygen sensitivity of [FeFe]-hydrogenase: a comparative study of active site mimics inside vs. outside the enzyme.

[FeFe]-hydrogenase is nature's most efficient proton reducing and H2-oxidizing enzyme. However, biotechnological applications are hampered by the O2 sensitivity of this metalloenzyme, and the mechanism of aerobic deactivation is not well understood. Here, we explore the oxygen sensitivity of four mimics of the organometallic active site cofactor of [FeFe]-hydrogenase, [Fe2(adt)(CO)6-x(CN)x]x- and [Fe2(pdt)(CO)6-x(CN)x]x- (x = 1, 2) as well as the corresponding cofactor variants of the enzyme by means of infrared, Mössbauer, and NMR spectroscopy. Additionally, we describe a straightforward synthetic recipe for the active site precursor complex Fe2(adt)(CO)6. Our data indicate that the aminodithiolate (adt) complex, which is the synthetic precursor of the natural active site cofactor, is most oxygen sensitive. This observation highlights the significance of proton transfer in aerobic deactivation, and supported by DFT calculations facilitates an identification of the responsible reactive oxygen species (ROS). Moreover, we show that the ligand environment of the iron ions critically influences the reactivity with O2 and ROS like superoxide and H2O2 as the oxygen sensitivity increases with the exchange of ligands from CO to CN-. The trends in aerobic deactivation observed for the model complexes are in line with the respective enzyme variants. Based on experimental and computational data, a model for the initial reaction of [FeFe]-hydrogenase with O2 is developed. Our study underscores the relevance of model systems in understanding biocatalysis and validates their potential as important tools for elucidating the chemistry of oxygen-induced deactivation of [FeFe]-hydrogenase.

An in vitro reconstitution system to monitor iron transfer to the active site during the maturation of [NiFe]-hydrogenase.

[NiFe]-hydrogenases (Hyds) comprise a small and a large subunit. The latter harbors the biologically unique [NiFe](CN)2CO active-site cofactor. The maturation process includes the assembly of the [Fe](CN)2CO cofactor precursor, nickel binding, endoproteolytic cleavage of the large subunit, and dimerization with the small subunit to yield active enzyme. The biosynthesis of the [Fe](CN)2CO moiety of [NiFe]-Hyd-1 and Hyd-2 occurs on the scaffold complex HybG-HypD (GD), whereas the HypC-HypD complex is specific for the assembly of Hyd-3. The metabolic source and the route for delivering iron to the active site remain unclear. To investigate the maturation process of O2-tolerant Hyd-1 from Escherichia coli, we developed an enzymatic in vitro reconstitution system that allows for the synthesis of Hyd-1 using only purified components. Together with this in vitro reconstitution system, we employed biochemical analyses, infrared spectroscopy (attenuated total reflection FTIR), mass spectrometry (MS), and microscale thermophoresis to monitor the iron transfer during the maturation process and to understand how the [Fe](CN)2CO cofactor precursor is ultimately incorporated into the large subunit. We demonstrate the direct transfer of iron from 57Fe-labeled GD complex to the large subunit of Hyd-1. Our data reveal that the GD complex exclusively interacts with the large subunit of Hyd-1 and Hyd-2 but not with the large subunit of Hyd-3. Furthermore, we show that the presence of iron in the active site is a prerequisite for nickel insertion. Taken together, these findings reveal how the [Fe](CN)2CO cofactor precursor is transferred and incorporated into the active site of [NiFe]-Hyd.

A personal account on 25 years of scientific literature on [FeFe]-hydrogenase.

[FeFe]-hydrogenases are gas-processing metalloenzymes that catalyze H2 oxidation and proton reduction (H2 release) in microorganisms. Their high turnover frequencies and lack of electrical overpotential in the hydrogen conversion reaction has inspired generations of biologists, chemists, and physicists to explore the inner workings of [FeFe]-hydrogenase. Here, we revisit 25 years of scientific literature on [FeFe]-hydrogenase and propose a personal account on 'must-read' research papers and review article that will allow interested scientists to follow the recent discussions on catalytic mechanism, O2 sensitivity, and the in vivo synthesis of the active site cofactor with its biologically uncommon ligands carbon monoxide and cyanide. Focused on-but not restricted to-structural biology and molecular biophysics, we highlight future directions that may inspire young investigators to pursue a career in the exciting and competitive field of [FeFe]-hydrogenase research.

Cyanide Binding to [FeFe]-Hydrogenase Stabilizes the Alternative Configuration of the Proton Transfer Pathway.

Hydrogenases are H2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN- ) ligands. Extrinsic CO and CN- , however, inhibit hydrogenases. The mechanism by which CN- binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN- -treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Šallowed us to distinguish intrinsic CN- and CO ligands and to show that extrinsic CN- binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN- treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally.

Proton Transfer Mechanisms in Bimetallic Hydrogenases.

Hydrogenases are metalloenzymes that catalyze proton reduction and H2 oxidation with outstanding efficiency. They are model systems for bioinorganic chemistry, including low-valent transition metals, hydride chemistry, and proton-coupled electron transfer. In this Account, we describe how photochemistry and infrared difference spectroscopy can be used to identify the dynamic hydrogen-bonding changes that facilitate proton transfer in [NiFe]- and [FeFe]-hydrogenase.[NiFe]-hydrogenase binds a heterobimetallic nickel/iron site embedded in the protein by four cysteine ligands. [FeFe]-hydrogenase carries a homobimetallic iron/iron site attached to the protein by only a single cysteine. Carbon monoxide and cyanide ligands in the active site facilitate detailed investigations of hydrogenase catalysis by infrared spectroscopy because of their strong signals and redox-dependent frequency shifts. We found that specific redox-state transitions in [NiFe]- and [FeFe]-hydrogenase can be triggered by visible light to record extremely sensitive "light-minus-dark" infrared difference spectra monitoring key amino acid residues. As these transitions are coupled to protonation changes, our data allowed investigation of dynamic hydrogen-bonding changes that go well beyond the resolution of protein crystallography.In [NiFe]-hydrogenase, photolysis of the bridging hydride ligand in the Ni-C state was followed by infrared difference spectroscopy. Our data clearly indicate the formation of a protonated cysteine residue as well as hydrogen-bonding changes involving a glutamic acid residue and a "dangling water" molecule. These findings are in excellent agreement with crystallographic analyses of [NiFe]-hydrogenase. In [FeFe]-hydrogenase, an external redox dye was used to accumulate the Hred state. Infrared difference spectra indicate hydrogen-bonding changes involving two glutamic acid residues and a conserved arginine residue. While crystallographic analyses of [FeFe]-hydrogenase in the oxidized state failed to explain the rapid proton transfer because of a breach in the succession of residues, our findings facilitated a precise molecular model of discontinued proton transfer.Comparing both systems, our data emphasize the role of the outer coordination sphere in bimetallic hydrogenases: we suggest that protonation of a nickel-ligating cysteine in [NiFe]-hydrogenase causes the notable preference toward H2 oxidation. On the contrary, proton transfer in [FeFe]-hydrogenase involves an adjacent cysteine as a relay group, promoting both H2 oxidation and proton reduction. These observations may guide the design of organometallic compounds that mimic the catalytic properties of hydrogenases.

Trapping an Oxidized and Protonated Intermediate of the [FeFe]-Hydrogenase Cofactor under Mildly Reducing Conditions.

The H-cluster is the catalytic cofactor of [FeFe]-hydrogenase, a metalloenzyme that catalyzes the formation of dihydrogen (H2). The catalytic diiron site of the H-cluster carries two cyanide and three carbon monoxide ligands, making it an excellent target for IR spectroscopy. In previous work, we identified an oxidized and protonated H-cluster species, whose IR signature differs from that of the oxidized resting state (Hox) by a small but distinct shift to higher frequencies. This "blue shift" was explained by a protonation at the [4Fe-4S] subcomplex of the H-cluster. The novel species, denoted HoxH, was preferentially accumulated at low pH and in the presence of the exogenous reductant sodium dithionite (NaDT). When HoxH was reacted with H2, the hydride state (Hhyd) was formed, a key intermediate of [FeFe]-hydrogenase turnover. A recent publication revisited our protocol for the accumulation of HoxH in wild-type [FeFe]-hydrogenase, concluding that inhibition by NaDT decay products rather than cofactor protonation causes the spectroscopic "blue shift". Here, we demonstrate that HoxH formation does not require the presence of NaDT (or its decay products), but accumulates also with the milder reductants tris(2-carboxyethyl)phosphine, dithiothreitol, or ascorbic acid, in particular at low pH. Our data consistently suggest that HoxH is accumulated when deprotonation of the H-cluster is impaired, thereby preventing the regain of the oxidized resting state Hox in the catalytic cycle.

Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly.

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN- and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN- and CO ligands on the scaffold complex HypCD.

Following Electroenzymatic Hydrogen Production by Rotating Ring-Disk Electrochemistry and Mass Spectrometry*.

Gas-processing metalloenzymes are of interest to future bio- and bioinspired technologies. Of particular importance are hydrogenases and nitrogenases, which both produce molecular hydrogen (H2 ) from proton (H+ ) reduction. Herein, we report on the use of rotating ring-disk electrochemistry (RRDE) and mass spectrometry (MS) to follow the production of H2 and isotopes produced from deuteron (D+ ) reduction (HD and D2 ) using the [FeFe]-hydrogenase from Clostridium pasteurianum, a model hydrogen-evolving metalloenzyme. This facilitates enzymology studies independent of non-innocent chemical reductants. We anticipate that these approaches will be of value in resolving the catalytic mechanisms of H2 -producing metalloenzymes and the design of bioinspired catalysts for H2 production and N2 fixation.

[FeFe]-hydrogenase maturation: H-cluster assembly intermediates tracked by electron paramagnetic resonance, infrared, and X-ray absorption spectroscopy.

[FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe-4S] cubane cysteine linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt = NH(CH2S)2)·[FeFe]-hydrogenase apo-protein binding only the [4Fe-4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe]adt). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts. We combined electron paramagnetic resonance, Fourier-transform infrared, and X-ray absorption spectroscopy to characterize intermediates of H-cluster assembly as initiated by mixing of the apo-protein (HydA1) from the green alga Chlamydomonas reinhardtii with [2Fe]adt. The three methods consistently show rapid formation of a complete H-cluster in the oxidized, CO-inhibited state (Hox-CO) already within seconds after the mixing. Moreover, FTIR spectroscopy support a model in which Hox-CO formation is preceded by a short-lived Hred'-CO-like intermediate. Accumulation of Hox-CO was followed by CO release resulting in the slower conversion to the catalytically active state (Hox) as well as formation of reduced states of the H-cluster.

Temperature Dependence of Structural Dynamics at the Catalytic Cofactor of [FeFe]-hydrogenase.

[FeFe]-hydrogenases are nature's blueprint for efficient hydrogen turnover. Understanding their enzymatic mechanism may improve technological H2 fuel generation. The active-site cofactor (H-cluster) consists of a [4Fe-4S] cluster ([4Fe]H), cysteine-linked to a diiron site ([2Fe]H) carrying an azadithiolate (adt) group, terminal cyanide and carbon monoxide ligands, and a bridging carbon monoxide (µCO) in the oxidized protein (Hox). Recently, the debate on the structure of reduced H-cluster states was intensified by the assignment of new species under cryogenic conditions. We investigated temperature effects (4-280 K) in infrared (IR) and X-ray absorption spectroscopy (XAS) data of [FeFe]-hydrogenases using fit analyses and quantum-chemical calculations. IR data from our laboratory and literature sources were evaluated. At ambient temperatures, reduced H-cluster states with a bridging hydride (µH-, in Hred and Hsred) or with an additional proton at [4Fe]H (Hred') or at the distal iron of [2Fe]H (Hhyd) prevail. At cryogenic temperatures, these species are largely replaced by states that hold a µCO, lack [4Fe]H protonation, and bind an additional proton at the adt nitrogen (HredH+ and HsredH+). XAS revealed the atomic coordinate dispersion (i.e., the Debye-Waller parameter, 2σ2) of the iron-ligand bonds and Fe-Fe distances in the oxidized and reduced H-cluster. 2σ2 showed a temperature dependence typical for the so-called protein-glass transition, with small changes below ∼200 K and a pronounced increase above this "breakpoint". This behavior is attributed to the freezing-out of larger-scale anharmonic motions of amino acid side chains and water species. We propose that protonation at [4Fe]H as well as ligand rearrangement and µH- binding at [2Fe]H are impaired because of restricted molecular mobility at cryogenic temperatures so that protonation can be biased toward adt. We conclude that a H-cluster with a µCO, selective [4Fe]H or [2Fe]H protonation, and catalytic proton transfer via adt facilitates efficient H2 conversion in [FeFe]-hydrogenase.

The Molecular Proceedings of Biological Hydrogen Turnover.

Over the past two decades, the bioinorganic chemistry of hydrogenases has attracted much interest from basic and applied research. Hydrogenases are highly efficient metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen (H2) in all domains of life. Their iron- and nickel-based cofactors represent promising blueprints for the design of biomimetic, synthetic catalysts. In this Account, we address the molecular proceedings of hydrogen turnover with [FeFe]-hydrogenases. The active site cofactor of [FeFe]-hydrogenases ("H-cluster") comprises a unique diiron complex linked to a [4Fe-4S] cluster via a single cysteine. Since it was discovered that a synthetic analogue of the diiron site can be incorporated into apoprotein in vitro to yield active enzyme, significant progress has been made toward a comprehensive understanding of hydrogenase catalysis. The diiron site carries three to four carbon monoxide (CO) and two cyanide (CN-) ligands that give rise to intense infrared (IR) absorption bands. These bands are sensitive reporters of the electron density across the H-cluster, which can be addressed by infrared spectroscopy to follow redox and protonation changes at the cofactor. Synthetic variation of the metal-bridging dithiolate ligand at the diiron site, as well as site-directed mutagenesis of amino acids, provides access to the proton pathways toward the cofactor. Quantum chemical calculations are employed to specifically assign IR bands to vibrational modes of the diatomic ligands and yield refined H-cluster geometries. Here, we provide an overview of recent research on [FeFe]-hydrogenases with emphasis on experimental and computational IR studies. We describe advances in attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR) and protein film electrochemistry, as well as density functional theory (DFT) calculations. Key cofactor species are discussed in terms of molecular geometry, redox state, and protonation. Isotope editing is introduced as a tool to evaluate the cofactor geometry beyond the limits of protein crystallography. In particular, the role of proton-coupled electron transfer (PCET) in the generation of catalytically relevant redox species is addressed. We propose that site-selective protonation of the H-cluster biases surplus electrons either to the [4Fe-4S] cluster or to the diiron site. Protonation of the [4Fe-4S] cluster prevents premature reduction at the diiron site and stabilizes a reactive, terminal hydride. The observed H-cluster species are assigned to rapid H2 conversion or to reactions possibly involved in activity regulation and cellular H2 sensing. In the catalytic cycle of [FeFe]-hydrogenases, an H-cluster geometry is preserved that features a bridging CO ligand. PCET levels the redox potential for two steps of sequential cofactor reduction. The concept of consecutive PCET at a geometrically inert cofactor with tight control of electron and proton localization may inspire the design of a novel generation of biomimetic catalysts for the production of H2 as a fuel.

Differential Protonation at the Catalytic Six-Iron Cofactor of [FeFe]-Hydrogenases Revealed by 57Fe Nuclear Resonance X-ray Scattering and Quantum Mechanics/Molecular Mechanics Analyses.

[FeFe]-hydrogenases are efficient biological hydrogen conversion catalysts and blueprints for technological fuel production. The relations between substrate interactions and electron/proton transfer events at their unique six-iron cofactor (H-cluster) need to be elucidated. The H-cluster comprises a four-iron cluster, [4Fe4S], linked to a diiron complex, [FeFe]. We combined 57Fe-specific X-ray nuclear resonance scattering experiments (NFS, nuclear forward scattering; NRVS, nuclear resonance vibrational spectroscopy) with quantum-mechanics/molecular-mechanics computations to study the [FeFe]-hydrogenase HYDA1 from a green alga. Selective 57Fe labeling at only [4Fe4S] or [FeFe], or at both subcomplexes was achieved by protein expression with a 57Fe salt and in vitro maturation with a synthetic diiron site precursor containing 57Fe. H-cluster states were populated under infrared spectroscopy control. NRVS spectral analyses facilitated assignment of the vibrational modes of the cofactor species. This approach revealed the H-cluster structure of the oxidized state (Hox) with a bridging carbon monoxide at [FeFe] and ligand rearrangement in the CO-inhibited state (Hox-CO). Protonation at a cysteine ligand of [4Fe4S] in the oxidized state occurring at low pH (HoxH) was indicated, in contrast to bridging hydride binding at [FeFe] in a one-electron reduced state (Hred). These findings are direct evidence for differential protonation either at the four-iron or diiron subcomplex of the H-cluster. NFS time-traces provided Mössbauer parameters such as the quadrupole splitting energy, which differ among cofactor states, thereby supporting selective protonation at either subcomplex. In combination with data for reduced states showing similar [4Fe4S] protonation as HoxH without (Hred') or with (Hhyd) a terminal hydride at [FeFe], our results imply that coordination geometry dynamics at the diiron site and proton-coupled electron transfer to either the four-iron or the diiron subcomplex discriminate catalytic and regulatory functions of [FeFe]-hydrogenases. We support a reaction cycle avoiding diiron site geometry changes during rapid H2 turnover.

Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations.

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN-) ligands. Hydrogen (H2) and oxygen (O2) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. 57Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fed in a [4Fe4S-2Fe]3- complex with the net spin on Fed whereas Hox-CO shows an apical CN- at Fed in a [4Fe4S-2Fe(CO)]3- complex with net spin sharing among Fep and Fed (proximal or distal iron ions in [2Fe]). At ambient O2 pressure, a novel H-cluster species (Hox-O2) accumulated in C169A, assigned to a [4Fe4S-2Fe(O2)]3- complex with an apical superoxide (O2-) carrying the net spin bound at Fed. H2 exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]3- complex with the net spin on the reduced cubane, an apical hydride at Fed, and a proton at a cysteine ligand. Hox-O2 and Hhyd are stabilized by impaired O2- protonation or proton release after H2 cleavage due to interruption of the proton path towards and out of the active site.

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H2). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN)2CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN)2CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase.

Protonation/reduction dynamics at the [4Fe-4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases.

The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN-) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN- vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN- infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

Spectroscopical Investigations on the Redox Chemistry of [FeFe]-Hydrogenases in the Presence of Carbon Monoxide.

[FeFe]-hydrogenases efficiently catalyzes hydrogen conversion at a unique [4Fe⁻4S]-[FeFe] cofactor, the so-called H-cluster. The catalytic reaction occurs at the diiron site, while the [4Fe⁻4S] cluster functions as a redox shuttle. In the oxidized resting state (Hox), the iron ions of the diiron site bind one cyanide (CN−) and carbon monoxide (CO) ligand each and a third carbonyl can be found in the Fe⁻Fe bridging position (µCO). In the presence of exogenous CO, A fourth CO ligand binds at the diiron site to form the oxidized, CO-inhibited H-cluster (Hox-CO). We investigated the reduced, CO-inhibited H-cluster (Hred´-CO) in this work. The stretching vibrations of the diatomic ligands were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR). Density functional theory (DFT) at the TPSSh/TZVP level was employed to analyze the cofactor geometry, as well as the redox and protonation state of the H-cluster. Selective 13CO isotope editing, spectro-electrochemistry, and correlation analysis of IR data identified a one-electron reduced, protonated [4Fe⁻4S] cluster and an apical CN− ligand at the diiron site in Hred´-CO. The reduced, CO-inhibited H-cluster forms independently of the sequence of CO binding and cofactor reduction, which implies that the ligand rearrangement at the diiron site upon CO inhibition is independent of the redox and protonation state of the [4Fe⁻4S] cluster. The relation of coordination dynamics to cofactor redox and protonation changes in hydrogen conversion catalysis and inhibition is discussed.

Bridging Hydride at Reduced H-Cluster Species in [FeFe]-Hydrogenases Revealed by Infrared Spectroscopy, Isotope Editing, and Quantum Chemistry.

[FeFe]-Hydrogenases contain a H2-converting cofactor (H-cluster) in which a canonical [4Fe-4S] cluster is linked to a unique diiron site with three carbon monoxide (CO) and two cyanide (CN-) ligands (e.g., in the oxidized state, Hox). There has been much debate whether reduction and hydrogen binding may result in alternative rotamer structures of the diiron site in a single (Hred) or double (Hsred) reduced H-cluster species. We employed infrared spectro-electrochemistry and site-selective isotope editing to monitor the CO/CN- stretching vibrations in [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. Density functional theory calculations yielded vibrational modes of the diatomic ligands for conceivable H-cluster structures. Correlation analysis of experimental and computational IR spectra has facilitated an assignment of Hred and Hsred to structures with a bridging hydride at the diiron site. Pronounced ligand rotation during µH binding seems to exclude Hred and Hsred as catalytic intermediates. Only states with a conservative H-cluster geometry featuring a µCO ligand are likely involved in rapid H2 turnover.

Retraction: The reductive phase of Rhodobacter sphaeroides cytochrome c oxidase disentangled by CO ligation.

Retraction of 'The reductive phase of Rhodobacter sphaeroides cytochrome c oxidase disentangled by CO ligation' by Hendrik Mohrmann et al., Phys. Chem. Chem. Phys., 2017, DOI: .