Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O₂ and CO

Abstract

The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H2-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H2. The H2 oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O2 and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site "gate" gas access, preventing molecules larger than H2 interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O2 is in fact an inhibitor of H2 oxidation by the RH, and that both H(+) reduction and H2 oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O2 arises due to interaction of O2 with the active site. Using protein film infrared electrochemistry (PFIRE) under H2 oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases.

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

“…41,42 Observation of a stable Ni a -L state under “physiologically relevant” conditions is therefore not a prerequisite for involvement of Ni a -L in the catalytic cycle. This could have implications for the catalytic relevance of Ni a -L in O 2 -sensitive Group 1 hydrogenases, and the Group 2 RH from R. eutropha , where Ni a -L species have so far only been observed following photolysis of Ni a -C. 1,40,82 …”

“…Despite the fact that solution assays on this enzyme demonstrated catalytic H 2 oxidation (albeit with a very low turnover frequency), the Ni a -R state(s) were not observed upon treatment with H 2 , 82,83,87 or electrochemical reduction in solution. 86 This lead to suggestion of a distinct two-state catalytic cycle for this enzyme involving only Ni a -SI and Ni a -C. 1,3 Recently, however, PFIRE spectra have revealed the presence of at least one Ni a -R state during catalytic H 2 oxidation, 40 suggesting that a common set of redox levels is conserved across the [NiFe] hydrogenases from quite different Groups.…”

“…The Group 2 [NiFe] hydrogenases are cytosolic and include the sensing hydrogenases, of which the regulatory hydrogenase (RH) from R. eutropha has been most studied. 3,40,52,82−87 R. eutropha RH has been shown to have a particularly low potential proximal cluster, below ca. −0.5 V, 52 and this may be significant in its behavior as this means the proximal cluster potential lies more negative than the potential of the H + /H 2 couple potential at neutral pH.…”

Controlling Redox Enzyme Orientation at Planar Electrodes

“…In addition, homogeneous distribution and orientation of enzymes in the 3D network is rarely achieved. Nonetheless, it is worth mentioning the original work of Vincent's group, who set up a Protein Film Infrared Electrochemistry (PFIRE) method for the monitoring of the enzyme conformation entrapped in carbon black particles [55]. Mazurenko et al also reported the resolution of enzyme partitions in carbon felts [21].…”

Infrared Microscopy as a Probe of Composition within a Model Biofuel Cell Electrode Prepared from Trametes versicolor Laccase

“…The optical properties of carbon at mid‐infrared wavelengths can cause spectral band distortions and limit radiation throughput . Recently, a strategy based on attenuated total reflection (ATR) infrared sampling was shown to provide valuable insights into bioelectrocatalytic pathways for enzymes adsorbed to high surface area carbon . Excellent sensitivity was achieved toward ligands at the catalytic active site in measurements on films of a model enzyme system cast onto the ATR crystal …”