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.

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal-Dependent Formate Dehydrogenase.

Biological carbon dioxide (CO2 ) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.

Anion Binding and Oxidative Modification at the Molybdenum Cofactor of Formate Dehydrogenase from Rhodobacter capsulatus Studied by X-ray Absorption Spectroscopy.

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N3-) or cyanate (OCN-). Azide provided best protection against O2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.

Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli.

The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors.

Functional Studies on Oligotropha carboxidovorans Molybdenum-Copper CO Dehydrogenase Produced in Escherichia coli.

The Mo/Cu-dependent CO dehydrogenase (CODH) from Oligotropha carboxidovorans is an enzyme that is able to catalyze both the oxidation of CO to CO2 and the oxidation of H2 to protons and electrons. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of O. carboxidovorans CODH in Escherichia coli. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H2 oxidation of Mo/Cu-dependent CODHs in the future.

Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamine N-Oxide Reductase.

The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.

[FeFe]-hydrogenase maturation.

Hydrogenases are metalloenzymes that catalyze the reversible reduction of protons at unusual metal centers. This Current Topic discusses recent advances in elucidating the steps involved in the biosynthesis of the complex metal cluster at the [FeFe]-hydrogenase (HydA) active site, known as the H-cluster. The H-cluster is composed of a 2Fe subcluster that is anchored within the active site by a bridging cysteine thiolate to a [4Fe-4S] cubane. The 2Fe subcluster contains carbon monoxide, cyanide, and bridging dithiolate ligands. H-cluster biosynthesis is now understood to occur stepwise; standard iron-sulfur cluster assembly machinery builds the [4Fe-4S] cubane of the H-cluster, while three specific maturase enzymes known as HydE, HydF, and HydG assemble the 2Fe subcluster. HydE and HydG are both radical S-adenosylmethionine enzymes that interact with an iron-sulfur cluster binding GTPase scaffold, HydF, during the construction of the 2Fe subcluster moiety. In an unprecedented biochemical reaction, HydG cleaves tyrosine and decomposes the resulting dehydroglycine into carbon monoxide and cyanide ligands. The role of HydE in the biosynthetic pathway remains undefined, although it is hypothesized to be critical for the synthesis of the bridging dithiolate. HydF is the site where the complete 2Fe subcluster is formed and ultimately delivered to the immature hydrogenase protein in the final step of [FeFe]-hydrogenase maturation. This work addresses the roles of and interactions among HydE, HydF, HydG, and HydA in the formation of the mature [FeFe]-hydrogenase.

Reversible H atom abstraction catalyzed by the radical S-adenosylmethionine enzyme HydG.

The organometallic H-cluster at the active site of [FeFe]-hydrogenases is synthesized by three accessory proteins, two of which are radical S-adenosylmethionine enzymes (HydE, HydG) and one of which is a GTPase (HydF). In this work we probed the specific role of H atom abstraction in HydG-catalyzed carbon monoxide and cyanide production from tyrosine. The isotope distributions of 5'-deoxyadenosine and p-cresol were evaluated using deuterium-labeled tyrosine substrates in H2O and D2O. The observation of multiply deuterated 5'-deoxyadenosine and deuterated S-adenosylmethionine when the reaction is carried out in D2O provides evidence for a 5'-deoxyadenosyl radical-mediated abstraction of a hydrogen atom from a solvent-exchangeable position as a reversible event.

H-cluster assembly during maturation of the [FeFe]-hydrogenase.

The organometallic H-cluster at the active site of the [FeFe]-hydrogenase serves as the site of reversible binding and reduction of protons to produce H2. The H-cluster is unique in biology, and consists of a 2Fe subcluster tethered to a typical [4Fe-4S] cluster by a single cysteine ligand. The remaining ligands to the 2Fe subcluster include three carbon monoxides, two cyanides, and a dithiomethylamine. This mini-review will focus on the significant advances in recent years in understanding the pathway for H-cluster biosynthesis, as well as the structures, roles, and mechanisms of the three enzymes directly involved.

Redox potentials elucidate the electron transfer pathway of NAD+-dependent formate dehydrogenases.

Metal-dependent, nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs) are complex metalloenzymes coupling biochemical transformations through intricate electron transfer pathways. Rhodobacter capsulatus FDH is a model enzyme for understanding coupled catalysis, in that reversible CO2 reduction and formate oxidation are linked to a flavin mononuclotide (FMN)-bound diaphorase module via seven iron-sulfur (FeS) clusters as a dimer of heterotetramers. Catalysis occurs at a bis-metal-binding pterin (Mo) binding two molybdopterin guanine dinucleotides (bis-MGD), a protein-based Cys residue and a participatory sulfido ligand. Insights regarding the proposed electron transfer mechanism between the bis-MGD and the FMN have been complicated by the discovery that an alternative pathway might occur via intersubunit electron transfer between two [4Fe4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the FeS clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven FeS clusters could be assigned. Furthermore, substitution of the active site residue Lys295 with Ala resulted in altered enzyme kinetics, primarily due to a more negative redox potential of the A1 [4Fe4S] cluster. Finally, characterization of the monomeric FdsGBAD heterotetramer exhibited slightly decreased formate oxidation activity and similar iron-sulfur clusters reduced relative to the dimeric heterotetramer. Comparison of the measured redox potentials relative to structurally defined FeS clusters support a mechanism by which electron transfer occurs within a heterotetrameric unit, with the interfacial [4Fe4S] cluster serving as a structural component toward the integrity of the heterodimeric structure to drive efficient catalysis.

Biochemical and kinetic characterization of radical S-adenosyl-L-methionine enzyme HydG.

The radical S-adenosyl-L-methionine (AdoMet) enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase H-cluster assembly. It catalyzes L-tyrosine cleavage to yield the H-cluster cyanide and carbon monoxide ligands as well as p-cresol. Clostridium acetobutylicum HydG contains the conserved CX3CX2C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX2CX22C motif proposed to coordinate a second [4Fe-4S] cluster. To improve our understanding of the roles of each of these iron-sulfur clusters in catalysis, we have generated HydG variants lacking either the N- or C-terminal cluster and examined these using spectroscopic and kinetic methods. We have used iron analyses, UV-visible spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant that cannot coordinate the radical AdoMet cluster to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (ΔCTD) harboring only the N-terminal cluster demonstrates that both clusters have similar UV-visible and EPR spectral properties, but that AdoMet binding and cleavage occur only at the N-terminal radical AdoMet cluster. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, we compared the Michaelis-Menten constants for AdoMet and L-tyrosine for reconstituted wild-type, C386S, and ΔCTD HydG and demonstrate that these C-terminal modifications do not affect the affinity for AdoMet but that the affinity for L-tyrosine is drastically reduced compared to that of wild-type HydG. Further detailed kinetic characterization of these HydG mutants demonstrates that the C-terminal cluster and residues are not essential for L-tyrosine cleavage to p-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO.

Radical AdoMet enzymes in complex metal cluster biosynthesis.

Radical S-adenosylmethionine (AdoMet) enzymes comprise a large superfamily of proteins that engage in a diverse series of biochemical transformations through generation of the highly reactive 5'-deoxyadenosyl radical intermediate. Recent advances into the biosynthesis of unique iron-sulfur (FeS)-containing cofactors such as the H-cluster in [FeFe]-hydrogenase, the FeMo-co in nitrogenase, as well as the iron-guanylylpyridinol (FeGP) cofactor in [Fe]-hydrogenase have implicated new roles for radical AdoMet enzymes in the biosynthesis of complex inorganic cofactors. Radical AdoMet enzymes in conjunction with scaffold proteins engage in modifying ubiquitous FeS precursors into unique clusters, through novel amino acid decomposition and sulfur insertion reactions. The ability of radical AdoMet enzymes to modify common metal centers to unusual metal cofactors may provide important clues into the stepwise evolution of these and other complex bioinorganic catalysts. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

HydG, the "dangler" iron, and catalytic production of free CO and CN-: implications for [FeFe]-hydrogenase maturation.

The organometallic H-cluster of the [FeFe]-hydrogenase consists of a [4Fe-4S] cubane bridged via a cysteinyl thiolate to a 2Fe subcluster ([2Fe]H) containing CO, CN-, and dithiomethylamine (DTMA) ligands. The H-cluster is synthesized by three dedicated maturation proteins: the radical SAM enzymes HydE and HydG synthesize the non-protein ligands, while the GTPase HydF serves as a scaffold for assembly of [2Fe]H prior to its delivery to the [FeFe]-hydrogenase containing the [4Fe-4S] cubane. HydG uses l-tyrosine as a substrate, cleaving it to produce p-cresol as well as the CO and CN- ligands to the H-cluster, although there is some question as to whether these are formed as free diatomics or as part of a [Fe(CO)2(CN)] synthon. Here we show that Clostridium acetobutylicum (C.a.) HydG catalyzes formation of multiple equivalents of free CO at rates comparable to those for CN- formation. Free CN- is also formed in excess molar equivalents over protein. A g = 8.9 EPR signal is observed for C.a. HydG reconstituted to load the 5th "dangler" iron of the auxiliary [4Fe-4S][FeCys] cluster and is assigned to this "dangler-loaded" cluster state. Free CO and CN- formation and the degree of activation of [FeFe]-hydrogenase all occur regardless of dangler loading, but are increased 10-35% in the dangler-loaded HydG; this indicates the dangler iron is not essential to this process but may affect relevant catalysis. During HydG turnover in the presence of myoglobin, the g = 8.9 signal remains unchanged, indicating that a [Fe(CO)2(CN)(Cys)] synthon is not formed at the dangler iron. Mutation of the only protein ligand to the dangler iron, H272, to alanine nearly completely abolishes both free CO formation and hydrogenase activation, however results show this is not due solely to the loss of the dangler iron. In experiments with wild type and H272A HydG, and with different degrees of dangler loading, we observe a consistent correlation between free CO/CN- formation and hydrogenase activation. Taken in full, our results point to free CO/CN-, but not an [Fe(CO)2(CN)(Cys)] synthon, as essential species in hydrogenase maturation.

[FeFe]-hydrogenase maturation: HydG-catalyzed synthesis of carbon monoxide.

Biosynthesis of the unusual organometallic H-cluster at the active site of the [FeFe]-hydrogenase requires three accessory proteins, two of which are radical AdoMet enzymes (HydE, HydG) and one of which is a GTPase (HydF). We demonstrate here that HydG catalyzes the synthesis of CO using tyrosine as a substrate. CO production was detected by using deoxyhemoglobin as a reporter and monitoring the appearance of the characteristic visible spectroscopic features of carboxyhemoglobin. Assays utilizing (13)C-tyrosine were analyzed by FTIR to confirm the production of HbCO and to demonstrate that the CO product was synthesized from tyrosine. CO ligation is a common feature at the active sites of the [FeFe], [NiFe], and [Fe]-only hydrogenases; however, this is the first report of the enzymatic synthesis of CO in hydrogenase maturation.

Cryo-EM structures reveal intricate Fe-S cluster arrangement and charging in Rhodobacter capsulatus formate dehydrogenase.

Metal-containing formate dehydrogenases (FDH) catalyse the reversible oxidation of formate to carbon dioxide at their molybdenum or tungsten active site. They display a diverse subunit and cofactor composition, but structural information on these enzymes is limited. Here we report the cryo-electron microscopic structures of the soluble Rhodobacter capsulatus FDH (RcFDH) as isolated and in the presence of reduced nicotinamide adenine dinucleotide (NADH). RcFDH assembles into a 360 kDa dimer of heterotetramers revealing a putative interconnection of electron pathway chains. In the presence of NADH, the RcFDH structure shows charging of cofactors, indicative of an increased electron load.