| Summary: | <p>This thesis investigates the catalytic mechanism of NiFe hydrogenases. The specific enzyme studied in this work, <em>E. coli</em> Hyd-1, is an efficient catalyst for H<sub>2</sub> oxidation even in the presence of O<sub>2</sub>. A method for studying the chemistry of the active site of this enzyme, under catalytic conditions, is developed. The combination of IR spectroscopy with protein film electrochemistry <em>in situ</em> is demonstrated. This was achieved by adsorbing the hydrogenase on a high surface area carbon nanoparticle electrode; and by the design of a spectroelectrochemical flow cell that provides efficient mass transport conditions.</p> <p>A complete redox characterisation of the active site for a hydrogenase immobilised on a carbon electrode is described for the first time. The study of the effect of pH on the distribution of redox states demonstrates the existence of a pH equilibrium between the Ni-C and the Ni-L states. It is shown that the active site responds to the pH of the external solution, and that the increase in pH acts as a driving force that removes the proton further away from the active site. Studies under electrocatalytic conditions provides direct evidence of intermediates of the catalytic cycle. The role of Ni-SI, Ni-R, and Ni-C is confirmed. Furthermore, Ni-L is detected under turnover conditions and therefore shown to be an important intermediate in the cycle. The detection of different protonation states of Ni-L and Ni-R is proposed to provide information on the transport of the protons as they start to move away from the active site.</p> <p>In the investigation of O<sub>2</sub> inhibition, Ni-B (detected spectroscopically) is directly related to the loss in activity upon the attack of O<sub>2</sub> for the first time. Also, the formation of solely Ni-B from the reaction with O<sub>2</sub> (no other O<sub>2</sub>-damaged species are detected) provides further evidence on the ability of O<sub>2</sub>-tolerant hydrogenases of having an effective mechanism for dealing with O<sub>2</sub> tolerance. A thorough study on the interaction of CO with Hyd-1 proves unequivocally that this O<sub>2</sub>-tolerant hydrogenase does bind CO, and that CO does inhibit its catalytic activity (both H2 and H+ reduction). This helps clarify how CO interacts with O<sub>2</sub>-tolerant hydrogenases.</p> <p>Overall, the work in this thesis contributed to the understanding of key mechanistic aspects in O<sub>2</sub>-tolerant hydrogenases. The technique for combining protein film electrochemistry with IR spectroscopy <em>in situ</em> shall provide valuable opportunities for providing new insight into the mechanisms of hydrogenases and other metalloenzymes that bind small molecules.</p>
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