The ubiquinone pool and ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) are central to cellular energy conversion in a very broad range of biological systems. Cytochrome bc1 converts oxidation-reduction (redox) free energy stored in the substrates ubiquinol and cytochrome c into a transmembrane electric potential and pH gradient essential for cellular maintenance and, because of its reversibility, mitochondrial regulation. However, the Qo site of primary energy conversion is poorly defined in crystal structures and experimentally difficult to access. We use molecular biology to excise, thermodynamically inactivate or sterically hinder redox cofactors of the high and low potential chains that meet at the Qo site to gain spectroscopic and electrochemical access to the molecular mechanisms of the cytochrome bc1. These cofactor 'knockouts' in the light-activatable photosynthetic bacterium Rhodobacter capsulatus selectively restrict the Qo site to just one turnover, securing single electron distributions in the redox cofactor chains. Double knockouts permit sole focus on the primary reactants at the Qo site. Novel measurements of events leading up to and including access to the Qo site are now a viable prospect. These include protein film voltammetry of cytochrome bc1 to activate and observe electron transfer through cofactor chains, to determine proton/electron coupled chemistry, and to clarify subunit motion. Infrared (ATR-FTIR) spectroscopy will be used to provide a more direct way of observing redox chemistry of cofactors and Qo site occupants, and their functional involvement with critical amino acids involved with setting potentials, proton exchange and structural change. EPR will examine paramagnetic states and radicals. Each of these methods, plus a novel use of NMR, will be used to help determine the number of ubiquinone occupants of the Qo site. Experiment aided by general application of electron tunneling expressions will explore the underlying engineering of the cytochrome bc1, including the effect of its dimeric structure, for normal operation in forward and reverse modes as well as determining the thresholds of failure and when deleterious side reactions, including damaging superoxide generation, become significant. These approaches naturally lend themselves to suggest concurrent systematic alterations in the chemistry and energetics of individual reactions by environmental change, cofactor replacement and further mutation to help identify mechanism at the Qo site. All aspects of the work are relevant to strategies for the development of drugs that can discriminate between host cytochromes bc1 and those of pathogens. As advances are made we will extend techniques to cytochrome oxidase and other oxidoreductases.
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| Zheng, Zhong; Dutton, P Leslie; Gunner, M R (2010) The measured and calculated affinity of methyl- and methoxy-substituted benzoquinones for the Q(A) site of bacterial reaction centers. Proteins 78:2638-54 |
| Chobot, Sarah E; Zhang, Haibo; Moser, Christopher C et al. (2008) Breaking the Q-cycle: finding new ways to study Qo through thermodynamic manipulations. J Bioenerg Biomembr 40:501-7 |
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| Osyczka, Artur; Zhang, Haibo; Mathe, Christelle et al. (2006) Role of the PEWY glutamate in hydroquinone-quinone oxidation-reduction catalysis in the Qo Site of cytochrome bc1. Biochemistry 45:10492-503 |
| Zhang, Haibo; Osyczka, Artur; Moser, Christopher C et al. (2006) Resilience of Rhodobacter sphaeroides cytochrome bc1 to heme c1 ligation changes. Biochemistry 45:14247-55 |
| Moser, Christopher C; Farid, Tammer A; Chobot, Sarah E et al. (2006) Electron tunneling chains of mitochondria. Biochim Biophys Acta 1757:1096-109 |
| Iwaki, Masayo; Yakovlev, Gregory; Hirst, Judy et al. (2005) Direct observation of redox-linked histidine protonation changes in the iron-sulfur protein of the cytochrome bc1 complex by ATR-FTIR spectroscopy. Biochemistry 44:4230-7 |
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