| Sumario: | <p>Hydrogenases catalyse the interconversion of H<sub>2</sub> and H<sup>+</sup>. Protein Film Electrochemistry (PFE), a technique in which a redox enzyme is adsorbed directly onto an electrode, enables a detailed description of the catalytic function of these metalloenzymes to be obtained. Unlike small-molecule electrocatalysts, the hydrogenase active site is surrounded by a protein structure ensuring that it is relatively unperturbed by the electrode surface. In this thesis, PFE is used alongside mathematical modelling to explain differences between [NiFe]- and [FeFe]-hydrogenases, highlighting some important considerations for efficient, reversible electrocatalysis.</p> <p>This thesis probes the unusual reaction between [NiFe]-hydrogenases and cyanide. Through a detailed study utilising PFE, Electron Paramagnetic Resonance (EPR) and Attenuated Total Reflection Infrared spectroelectrochemistry (ATR-IR), it is demonstrated that cyanide promotes the formation of the inactive Ni-B state. Preferred formation of the Ni-B state over more slowly reactivating Unready states is considered an important characteristic of the O<sub>2</sub>-tolerant class of [NiFe]-hydrogenases.</p> <p>The nature of the Ni-L state, commonly thought to be an artefact formed when a [NiFe]-hydrogenase is exposed to visible light, is probed <em>via</em> EPR and ATR-IR. In this thesis, the Ni-L state is shown to occur in samples of Hydrogenase-1 from <em>Escherichia coli</em> that have not been exposed to visible light, calling into question the true nature of this state.</p> <p>Finally, this thesis details the first study in which PFE is used to investigate the spontaneous incorporation of a synthetic active site mimic complex into apo-hydrogenase. Incorporation into apo-hydrogenase from <em>Chlamydomonas reinhardtii</em> and <em>Clostridium pasteurianum</em> is discussed, in both cases resulting in fully functional [FeFe]-hydrogenase, electrochemically indistinguishable from the native enzyme.</p>
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