A novel in situ IR spectroscopic approach is demonstrated for the characterization of hydrogenase during catalytic turnover. E. coli hydrogenase 1 (Hyd-1) is adsorbed on a high surface-area carbon electrode and subjected to the same electrochemical control and efficient supply of substrate as in protein film electrochemistry during spectral acquisition. The spectra reveal that the active site state known as Ni-L, observed in other NiFe hydrogenases only under illumination or at cryogenic temperatures, can be generated reversibly in the dark at ambient temperature under both turnover and non-turnover conditions. The observation that Ni-L is present at all potentials during turnover under H2 suggests that the final steps in the catalytic cycle of H2 oxidation by Hyd-1 involve sequential proton and electron transfer via Ni-L. A broadly applicable IR spectroscopic technique is presented for addressing electrode-adsorbed redox enzymes under fast catalytic turnover.

Catalysis of H2 production and oxidation reactions is critical in renewable energy systems based around H2 as a clean fuel, but the present reliance on platinum-based catalysts is not sustainable. In nature, H2 is oxidized at minimal overpotential and high turnover frequencies at [NiFe] catalytic sites in hydrogenase enzymes. Although an outline mechanism has been established for the [NiFe] hydrogenases involving heterolytic cleavage of H2 followed by a first and then second transfer of a proton and electron away from the active site, details remain vague concerning how the proton transfers are facilitated by the protein environment close to the active site. Furthermore, although [NiFe] hydrogenases from different organisms or cellular environments share a common active site, they exhibit a broad range of catalytic characteristics indicating the importance of subtle changes in the surrounding protein in controlling their behavior. Here we review recent time-resolved infrared (IR) spectroscopic studies and IR spectroelectrochemical studies carried out in situ during electrocatalytic turnover. Additionally, we re-evaluate the significant body of IR spectroscopic data on hydrogenase active site states determined through more conventional solution studies, in order to highlight mechanistic steps that seem to apply generally across the [NiFe] hydrogenases, as well as steps which so far seem limited to specific groups of these enzymes. This analysis is intended to help focus attention on the key open questions where further work is needed to assess important aspects of proton and electron transfer in the mechanism of [NiFe] hydrogenases.

Despite extensive studies on [NiFe]-hydrogenases, the mechanism by which these enzymes produce and activate H2 so efficiently remains unclear. A well-known EPR-active state produced under H2 and known as Ni-C is assigned as a NiIII–FeII species with a hydrido ligand in the bridging position between the two metals. It has long been known that low-temperature photolysis of Ni-C yields distinctive EPR-active states, collectively termed Ni-L, that are attributed to migration of the bridging-H species as a proton; however, Ni-L has mainly been regarded as an artifact with no mechanistic relevance. It is now demonstrated, based on EPR and infrared spectroscopic studies, that the Ni-C to Ni-L interconversion in Hydrogenase-1 (Hyd-1) from Escherichia coli is a pH-dependent process that proceeds readily in the dark—proton migration from Ni-C being favored as the pH is increased. The persistence of Ni-L in Hyd-1 must relate to unassigned differences in proton affinities of metal and adjacent amino acid sites, although the unusually high reduction potentials of the adjacent Fe–S centers in this O2-tolerant hydrogenase might also be a contributory factor, impeding elementary electron transfer off the [NiFe] site after proton departure. The results provide compelling evidence that Ni-L is a true, albeit elusive, catalytic intermediate of [NiFe]-hydrogenases.

A novel in situ IR spectroscopic approach is demonstrated for the characterization of hydrogenase during catalytic turnover. E. coli hydrogenase 1 (Hyd-1) is adsorbed on a high surface-area carbon electrode and subjected to the same electrochemical control and efficient supply of substrate as in protein film electrochemistry during spectral acquisition. The spectra reveal that the active site state known as Ni-L, observed in other NiFe hydrogenases only under illumination or at cryogenic temperatures, can be generated reversibly in the dark at ambient temperature under both turnover and nonturnover conditions. The observation that Ni-L is present at all potentials during turnover under H 2 suggests that the final steps in the catalytic cycle of H 2 oxidation by Hyd-1 involve sequential proton and electron transfer via Ni-L. A broadly applicable IR spectroscopic technique is presented for addressing electrode-adsorbed redox enzymes under fast catalytic turnover.

We present a study of electrocatalysis by an enzyme adsorbed on a range of carbon materials, with different size, surface area, morphology and graphitic structure, which are either commercially available or prepared via simple, established protocols. We choose as our model enzyme the hydrogenase I from E. coli (Hyd-1), which is an active catalyst for H2 oxidation, is relatively robust and has been demonstrated in H2 fuel cells and H2-driven chemical synthesis. The carbon materials were characterised according to their surface area, surface morphology and graphitic character, and we use the electrocatalytic H2 oxidation current for Hyd-1 adsorbed on these materials to evaluate their effectiveness as enzyme electrodes. Here, we show that a variety of carbon materials are suitable for adsorbing hydrogenases in an electroactive configuration. This unified study provides insight into selection and design of carbon materials for study of redox enzymes and different applications of enzyme electrocatalysis.