Abstract:
The Rieske/cytochrome b complexes, also known as cytochrome bc complexes, catalyze a unique oxidant-induced reduction reaction at their quinol oxidase (Qo) sites, in which substrate hydroquinone reduces two distinct electron transfer chains, one leading to a series of high-potential electron carriers, the second to low-potential cytochrome b. This reaction is a critical step in energy storage by the Q-cycle. The semiquinone intermediate in this reaction can also reduce O2 to produce deleterious superoxide. It is yet unknown how the enzyme controls this reaction, though numerous models have been proposed. In previous work we were able to trap a Q-cycle semiquinone anion intermediate, termed SQo in bacterial cyt bc1 by rapid freeze-quenching. In this work, we apply pulsed EPR techniques to determine the location of SQo in mictochondrial complex and that mitochondrial SQo has highly unusual properties. In contrast with previous semiquinone intermediates, SQo is not thermodynamically stabilized, or even destabilized with respect to solution. It is localized in the Qo pocket at a niche, which is distinct from previously described inhibitor-binding sites, but is sufficiently close to cytochrome bL to allow rapid electron transfer. Both the location of the binding sites and EPR analysis show that SQo is not stabilized by hydrogen bonds to proteins. These results indicate that the formation of SQo involves "stripping" of both substrate protons during the initial oxidation to the high potential chain, as well as conformational changes of both semiquinone species and Qo site proteins components. The resulting charged radical is kinetically trapped, rather than thermodynamically stabilized (as in most enzymatic semiquinone species), maintaining redox energy to drive electron transfer to cytochrome bL, while minimizing certain Q-cycle bypass reactions including oxidation of pre-reduced cytochrome b and reduction of O2.
