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Abstract

Quinone molecules are ubiquitous in living organisms. They are found either within the lipid phase of the biological membrane (quinone pool) or are bound in specific binding sites within membrane-bound protein complexes. The biological function of such bound quinones is determined by their ability to be reduced and/or oxidized in two successive one-electron steps. As a result, quinones are involved as one- or two-electron donors or acceptors in a large number of biological electron-transfer steps occurring during respiratory or photosynthetic processes. The intermediate formed by a one-electron reduction step is a semiquinone, which is paramagnetic and can be studied by electron paramagnetic resonance (EPR) spectroscopy. Detailed studies of such states can provide important structural information on these intermediates in such electron-transfer processes. In this study, we focus on the redox-active ubiquinone-6 of the yeast cytochrome bc₁ complex (QCR, ubiquinol: cytochrome c oxidoreductase) from Saccharomyces cerevisiae at the so-called Q_i site. Although the location of the Q_i binding pocket is quite well known, details about its exact binding are less clear. Currently, three different X-ray crystallographic studies suggest three different binding geometries for Q_i. Recent studies in the bacterial system (Rhodobacter sphaeroides) have suggested a direct coordination to histidine as proposed in the chicken heart crystal structure model. Using the yeast system we apply EPR and especially relaxation filtered hyperfine (REFINE) spectroscopy to study the Q_i binding site. ¹⁴N-electron spin-echo envelope modulation spectroscopy together with an inversion-recovery filter (REFINE) is applied to resolve the question of whether ¹⁴N modulations arise from interactions to Q_i^·- or to the Rieske iron–sulphur center. These results are discussed with regard to the location and potential function of Q_i in the enzyme.