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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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Chemical modification of antibodies is critical for many research areas including therapeutic and biotechnological applications. In particular, strategies for site-specific chemical modification via non-natural amino acids forming homogenous immunoconjugates are of interest. The use of fully functional single-domain antibodies derived from naturally occurring heavy chain antibodies in Camelidae species is attractive due to their enhanced properties, which are discussed in this piece of work. In terms of chemical antibody modification, much of the existing research is focused on modification away from binding regions, thus minimising disturbance to antibody function. In this thesis however, modifications within the binding region of the single-domain antibody cAb-Lys3 are described in a site-specific fashion with the aim of modulating binding affinities. An efficient and high yielding method for the expression and purification of cAh-Lys3 is described, followed by the site-specific installation of dehydroalanine, an electrophilic non-natural amino acid, able to react with nucleophiles that are inert to reaction with other proteinogenic amino acids. Then, the use of dehydroalanine as a unique reaction handle is explored. Firstly, the addition of short alkyl chains to dehydroalanine within the binding region of cAb-Lys3 is described with the aim of increasing hydrophobic interactions when binding to antigen.

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