"Restricted: Full content available from 2014-07-1 (ISO 8601)". . "2012-05-02T10:05:02.977Z"^^. . . . "Hydrogenases are a class of metalloenzymes which catalyse H\u2082 oxidation and its reverse reaction, H\u207A reduction. There is interest in investigating how H\u2082 as an energy carrier is cycled in biology. Hydrogenases have also been studied extensively because there are potential applications for them as catalysts for H\u2082 oxidation in fuel cells or H\u2082 production via light-driven water splitting. For these applications, the ability for the hydrogenase to work in the presence of O\u2082 is an important issue. The microorganism Ralstonia eutropha is a well-studied model aerobic H\u2082 oxidiser: it can adopt H\u2082 as the sole energy source to grow cells in the presence of O\u2082. It produces at least three distinct O\u2082-tolerant NiFe hydrogenases: the membrane-bound hydrogenase (MBH), the NAD\u207A-reducing soluble hydrogenase (SH) and the regulatory hydrogenase (RH). This Thesis employs protein film electrochemistry (PFE) to study the SH and RH. It is found that the SH is able to work in both direction (H\u2082 oxidation and H\u207A reduction) with minimum overpotential, which is critical in coupling 2H\u207A/H\u2082 cycling with the closely spaced NAD\u207A/NADH potential. Reactions of the SH with O\u2082 have been investigated, revealing at least two distinct O\u2082-inactivated states, but consistent with the requirement for the SH to function in air, it can be reactivated in the presence of O\u2082 at low potentials which could be provided by the NAD\u207A/NADH pool in vivo. The affinity of the RH for H\u2082 was determined by PFE and found to be slightly higher than that of the SH and MBH. This may provide a way for the microbe to regulate hydrogenase expression in response to the H\u2082 availability. Carbon monoxide and O\u2082-inactivated states of the RH have been identified for the first time, confirming that a constricted gas channel is not sufficient to explain its O\u2082 tolerance. Observation of potential dependent reactions in hydrogenases means that it is important to have spectroscopic methods for characterising states triggered by inhibitors and potential. An Infrared spectroelectrochemical approach suitable for studying metalloenzymes has been developed and preliminary spectra on RH recorded. This method should provide many opportunities for future studies of redox states of hydrogenases.". "This thesis is not currently available in ORA.". "Vincent, K. A.". "Liu, Juan". . . . . . . "born digital". "mixed material". "thesis". "hydrogenase". "Inorganic chemistry". "infrared spectroelectrochemistry". "protein film electrochemistry". "Electrochemistry and electrolysis". "Physical Sciences". "urn:uuid:ed955b6f-1ed2-4e2e-a3b9-c53e39543947". "ora:6214". "ora:6214". . "en". . "fedoraAdmin". "2012". "Electrochemical and infrared spectroelectrochemical methods applied to the NiFe hydrogenases of Ralstonia eutropha". "2012-06-21T02:02:54.823Z"^^. .