| Summary: | This Thesis describes the development of a new approach to using IR spectroscopy to follow redox chemistry of metalloproteins. Incorporation of a high surface area carbon particle electrode into an attenuated total reflectance (ATR) geometry allows IR spectra to be recorded on enzymes under in situ electrochemical control over a wide potential range without the need for solution redox mediators. A spectroelectrochemical cell, built in-house, provides control of temperature, and solution and gas composition, enabling a range of reaction triggers. The ATR-IR spectroelectrochemical approach is used for determining the potential of a transition between redox states of the regulatory [NiFe] -hydrogenase from Ralstonia eutropha, an enzyme which has not been studied previously under electrochemical control. The technique is then applied to the 02-tolerant Hydrogenase 1 (Hyd-l) from Escherichia coli and used to collect redox titration data allowing the determination of midpoint potentials for transitions between four redox levels of the enzyme. A hydrogenase state known as Ni-L previously detected only under non-physiological conditions (illumination and/or cryogenic temperature) is observed in Hyd-l without illumination and at ambient temperature. Ni-L is thought to arise from deprotonation of Ni-C, a NiIII(H-)FeII active site state, giving NiIFeII. O~servation of Ni-L under ambient conditions suggests it is relevant to the catalytic cycle, and this is discussed in the context of the mechanism of H2 oxidation by [NiFe]-hydrogenases.
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