Akinsiku, Akinyemi Olutosin; (2001) Ubiquinone binding sites of mitochondrial bc1 complex. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The cytochrome bc1 complex is a central component of the electron-transfer respiratory chain in mitochondria. It couples electron transfer to proton translocation across the membrane by a Q-cycle mechanism. It contains two ubiquinone-binding sites (Qo, ubiquinol oxidation and Qi, ubiquinone reduction) which are good targets for pesticides. Although the mechanism of the Qo oxidation reaction has been much studied, the details remain controversial, in particular, the way in which electrons are bifurcated along two separate routes and whether the Qo site has to bind one or two ubiquinones in order to be catalytically active. Resolving these issues is essential for understanding the chemistry of this unique reaction and useful in the development of pesticides. This thesis describes studies of the occupancy state of the Qo site, redox-linked changes in amino acids and protein conformation and characterisation of the binding site of new inhibitors using steady state kinetics, redox potentiometry, and optical, EPR and FTIR spectroscopy. To address the question of the occupancy state of the Qo site, steady state kinetics was used to characterise the binding mode of the Qo site inhibitor MOA- stilbene in relation to the substrate decylubiquinol. This revealed a mixed type of inhibition, which shows that the previous conclusion of separate binding sites for MOA-stilbene and ubiquinone is questionable. A more direct method, EPR spectroscopy, which has proved to be highly sensitive to the degree and nature of the Qo site occupants (Q/QH2 or inhibitors), was also used. The results were consistent with a single occupancy model of the Qo site. To complement these studies, molecular modelling studies with the available crystal structure and data from mutagenesis studies to identify residues conferring resistance to inhibitors were used. In addition, an attempt was made to model two ubiquinone molecules into the Qo site. Finally, competition between two classes of Qo site inhibitors was also investigated. These studies were consistent with a single occupancy model of the Qo site as suggested from kinetic and EPR studies. FTIR difference spectroscopy in combination with selective photochemical reduction is a powerful technique for studying redox-linked changes in amino acids and protein conformation. The FTIR redox difference spectra of the components of bovine bc1 complex in native and inhibitor bound states were resolved. Signal assignments were aided by comparison to FTIR redox difference spectra of soluble domains of individual redox centres and model compounds. Inhibitors of the bc1 complex have been an important tool in the elucidation of the mechanism of this multi-protein complex and are of great commercial interest as pesticides. Characterisation of new inhibitors revealed that eight were Qo site inhibitors and the other two were Qi site inhibitors.

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