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Title: Studies of NADH oxidation by NADH : ubiquinone oxidoreductase (complex I) from bovine mitochondria
Author: Barker, C. D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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The work described here focuses primarily on the flavoprotein (Fp) subcomplexes of complex I, as a subject for studying NADH oxidation. Fp is the smallest catalytically active subcomplex of complex I. It contains only two subunits and three cofactors – the FMN and a [4Fe-4S] cluster bound by the 51 kDa subunit, and a [2Fe-2S] cluster bound by the 24 kDa subunit. First, the development of a reproducible method for isolating stable and catalytically active Fp from complex I, using chaotropic resolution and ion-exchange chromatography, is described. The subcomplex produced contains the two subunits in equal amounts, and both [Fe-S] clusters and the FMN were present. Fp was capable of NADH oxidation and electron transfer to artificial electron acceptors in standard solution assays. The potential of the FMN was measured and values agree well with those reported from electron paramagnetic resonance (EPR). NADH oxidation was observed and explained by a model which incorporates mass transport of substrate. Michaelis-Menten enzyme kinetics, and interfacial electron transfer. The apparent potential of the active site under non-turnover conditions was higher than the values measured in the absence of substrate, providing insights into the mechanism of NADH oxidation. Unexpectedly, the rate of NAD+ reduction was fastest at intermediate potential – as the driving force for the reaction was increased a sharp maximum in activity was observed, after which there was a marked decrease. Two mechanistic models are proposed and shown to reproduce the experimentally observed voltammograms. In a second investigation, the [Fe-S] clusters in complex I were studied by EPR spectroscopy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available