A [4Fe-4S]-Fe(CO)(CN)-L-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly.

Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5 : probing the roles of system-specific accessory proteins

A subset of bacterial [NiFe]-hydrogenases have been shown to be capable of activating dihydrogen-catalysis under aerobic conditions; however, it remains relatively unclear how the assembly and activation of these enzymes is carried out in the presence of air. Acquiring this knowledge is important if a generic method for achieving production of O2-resistant [NiFe]-hydrogenases within heterologous hosts is to be developed. Salmonella enterica serovar Typhimurium synthesizes the [NiFe]-hydrogenase-5 (Hyd-5) enzyme under aerobic conditions. As well as structural genes, the Hyd-5 operon also contains several accessory genes that are predicted to be involved in different stages of biosynthesis of the enzyme. In this work, deletions in the hydF, hydG, and hydH genes have been constructed. The hydF gene encodes a protein related to Ralstonia eutropha HoxO, which is known to interact with the small subunit of a [NiFe]-hydrogenase. HydG is predicted to be a fusion of the R. eutropha HoxQ and HoxR proteins, both of which have been implicated in the biosynthesis of an O2-tolerant hydrogenase, and HydH is a homologue of R. eutropha HoxV, which is a scaffold for [NiFe] cofactor assembly. It is shown here that HydG and HydH play essential roles in Hyd-5 biosynthesis. Hyd-5 can be isolated and characterized from a ΔhydF strain, indicating that HydF may not play the same vital role as the orthologous HoxO. This study, therefore, emphasises differences that can be observed when comparing the function of hydrogenase maturases in different biological systems

How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure

‘Oxygen-tolerant’ [NiFe]-hydrogenases can catalyze H(2) oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O(2) molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H(2) oxidation released at the active site of one partner are immediately transferred to its counterpart when an O(2) molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O(2)-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins

Enhanced oxygen-tolerance of the full heterotrimeric membrane-bound [NiFe]-hydrogenase of ralstonia eutropha.

Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen-oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane

Spectroscopic characterization of the key catalytic intermediate Ni-C in the O2-tolerant [NiFe] Hydrogenase I from Aquifex aeolicus: evidence of a weakly bound hydride.

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Reply to Mouesca et al.: Electronic structure of the proximal [4Fe-3S] cluster of O₂-tolerant [NiFe] hydrogenases

Mouesca et al. (1) criticize our analysis (2) of the superoxidized proximal [4Fe-3S]5+ cluster in O2-tolerant hydrogenases (A) that was also studied in Volbeda et al. (3), albeit from a different organism. Both analyses were based on broken-symmetry (BS) density functional theory (DFT) calculations (2, 3), which presently is the only feasible alternative for the treatment of complex systems like A. However, BS-DFT is a crude approach and we regard the differences in refs. 2 and 3 as subtle rather than major. The critique raised in Mouesca et al. (1) that our cluster model was too small and incorporated spurious constraints is unfounded. In Volbeda et al. (3), only three additional amino acid residues in the third coordination sphere were included, but their importance was not demonstrated. Furthermore, our constraints allowed for sufficient structural relaxation and the link atoms used in the quantum mechanical/molecular mechanical investigation (3) imposed almost identical constraints. The allegation that our selection of BS solution Ox2_24 over OX2_14 (favored in ref. 3) on the basis of “chemical intuition” and “weak isomer shift considerations” is factually wrong. Instead, we analyzed relative energies and four Mössbauer parameters (δ, ΔEQ, η, A) for each iron site, whereas in Volbeda et al. (3) only energetics and unsigned ΔEQ values were used. The energy difference in favor of OX2_14 (3) is below 2 kcal/mol, which is well within the uncertainty of BS-DFT. Thus, we have presented more solid evidence for our choice than the authors of ref. 3. Because our quantum mechanical model was not inferior to that of Volbeda et al. (3), we have no reason to revoke deprotonation of GLU82. Our choice was derived from a comprehensive comparison of a wide range of spectroscopic parameters, and no conclusive evidence against it was presented in Mouesca et al. (1). The authors of refs. 1 and 3 argue in favor of OX2_14 as leading to an explanation of the cluster’s reactivity. However, no sound conclusions on transition states or mechanisms can be drawn from the analysis of the magnetic properties of one reactant only. Thus, the argument is invalid. Mouesca et al. (1) argue that OX2_24 emerges from unrecognized, unwanted charge migration, which leads to “local spin state 3/2, instead of 5/2, for Fe2,” and therefore is an “artificially trapped electronic state.” We note that: (i) the magnetic properties are well explained by OX2_24; (ii) the reactivity argument is invalid; and (iii) the local spin is not an observable and BS-DFT does not conserve the total spin but only its projection onto the z axis. The assignment of oxidation states and local spins in refs. 1 and 3 relies on differences as small as 0.1 electrons in Mulliken spin populations. In our experience, differences of this order are not conclusive. Importantly, the lower spin population on Fe2 in OX2_24 is simply a result of spin-canting, as well as (partial) formation of covalent chemical bonds between spin-carrying fragments. Related effects are predominant in BS-DFT calculations on antiferromagnetic clusters. The claim that our OX2_24 has SFe2 = 3/2 is therefore unsupported and unrealistic. In conclusion, although we do not exclude future refinements of our understanding of the [4Fe-3S] cluster, the arguments brought forward in Mouesca et al. (1) are insufficient to invalidate our original analysis (2)

Electronic structure of the unique [4Fe-3S] cluster in O₂-tolerant hydrogenases characterized by ⁵⁷Fe Mössbauer and EPR spectroscopy

Iron–sulfur clusters are ubiquitous electron transfer cofactors in hydrogenases. Their types and redox properties are important for H2 catalysis, but, recently, their role in a protection mechanism against oxidative inactivation has also been recognized for a [4Fe-3S] cluster in O2-tolerant group 1 [NiFe] hydrogenases. This cluster, which is uniquely coordinated by six cysteines, is situated in the proximity of the catalytic [NiFe] site and exhibits unusual redox versatility. The [4Fe-3S] cluster in hydrogenase (Hase) I from Aquifex aeolicus performs two redox transitions within a very small potential range, forming a superoxidized state above +200 mV vs. standard hydrogen electrode (SHE). Crystallographic data has revealed that this state is stabilized by the coordination of one of the iron atoms to a backbone nitrogen. Thus, the proximal [4Fe-3S] cluster undergoes redox-dependent changes to serve multiple purposes beyond classical electron transfer. In this paper, we present field-dependent 57Fe-Mössbauer and EPR data for Hase I, which, in conjunction with spectroscopically calibrated density functional theory (DFT) calculations, reveal the distribution of Fe valences and spin-coupling schemes for the iron–sulfur clusters. The data demonstrate that the electronic structure of the [4Fe-3S] core in its three oxidation states closely resembles that of corresponding conventional [4Fe-4S] cubanes, albeit with distinct differences for some individual iron sites. The medial and distal iron–sulfur clusters have similar electronic properties as the corresponding cofactors in standard hydrogenases, although their redox potentials are higher