Re-engineering a NiFe hydrogenase to increase the H2 production bias while maintaining native levels of O2 tolerance

Naturally occurring oxygen tolerant NiFe membrane bound hydrogenases have a conserved catalytic bias towards hydrogen oxidation which limits their technological value. We present an Escherichia coli Hyd-1 amino acid exchange that apparently causes the catalytic rate of H2 production to double but does not impact the O2 tolerance

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

Probing intermediates in the activation cycle of [NiFe] hydrogenase by infrared spectroscopy: the Ni-SIr state and its light sensitivity

The [NiFe] hydrogenase from the sulphate-reducing bacterium Desulfovibrio vulgaris Miyazaki F is reversibly inhibited in the presence of molecular oxygen. A key intermediate in the reactivation process, Ni-SIr, provides the link between fully oxidized (Ni-A, Ni-B) and active (Ni-SIa, Ni-C and Ni-R) forms of hydrogenase. In this work Ni-SIr was found to be light-sensitive (T ≤ 110 K), similar to the active Ni-C and the CO-inhibited states. Transition to the final photoproduct state (Ni-SL) was shown to involve an additional transient light-induced state (Ni-SI1961). Rapid scan kinetic infrared measurements provided activation energies for the transition from Ni-SL to Ni-SIr in protonated as well as in deuterated samples. The inhibitor CO was found not to react with the active site of the Ni-SL state. The wavelength dependence of the Ni-SIr photoconversion was examined in the range between 410 and 680 nm. Light-induced effects were associated with a nickel-centred electronic transition, possibly involving a change in the spin state of nickel (Ni2+). In addition, at T ≤ 40 K the CN− stretching vibrations of Ni-SL were found to be dependent on the colour of the monochromatic light used to irradiate the species, suggesting a change in the interaction of the hydrogen-bonding network of the surrounding amino acids. A possible mechanism for the photochemical process, involving displacement of the oxygen-based ligand, is discussed

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