Electrochemical and infrared spectroelectrochemical methods applied to the NiFe hydrogenases of Ralstonia eutropha

Thesis

Hydrogenases are a class of metalloenzymes which catalyse H₂ oxidation and its reverse reaction, H⁺ reduction. There is interest in investigating how H₂ as an energy carrier is cycled in biology. Hydrogenases have also been studied extensively because there are potential applications for them as catalysts for H₂ oxidation in fuel cells or H₂ production via light-driven water splitting. For these applications, the ability for the hydrogenase to work in the presence of O₂ is an important issue. The microorganism Ralstonia eutropha is a well-studied model aerobic H₂ oxidiser: it can adopt H₂ as the sole energy source to grow cells in the presence of O₂. It produces at least three distinct O₂-tolerant NiFe hydrogenases: the membrane-bound hydrogenase (MBH), the NAD⁺-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 (H₂ oxidation and H⁺ reduction) with minimum overpotential, which is critical in coupling 2H⁺/H₂ cycling with the closely spaced NAD⁺/NADH potential. Reactions of the SH with O₂ have been investigated, revealing at least two distinct O₂-inactivated states, but consistent with the requirement for the SH to function in air, it can be reactivated in the presence of O₂ at low potentials which could be provided by the NAD⁺/NADH pool in vivo. The affinity of the RH for H₂ 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 H₂ availability. Carbon monoxide and O₂-inactivated states of the RH have been identified for the first time, confirming that a constricted gas channel is not sufficient to explain its O₂ 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.

Authors: Liu, J

• Publication date: 2012
• DOI: 10.5287/ora-eopojd8dn
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: Oxford University, UK
• Language: English
• Keywords: protein film electrochemistry; hydrogenase; infrared spectroelectrochemistry
• Subjects: Inorganic chemistry; Physical Sciences; Electrochemistry and electrolysis