A Caged, Destabilized, Free Radical Intermediate in the Q‐Cycle. Issue 14 (5th September 2013)

• Record Type: Journal Article
• Authors: Vennam, Preethi R.; Fisher, Nicholas; Krzyaniak, Matthew D.; Kramer, David M.; Bowman, Michael K.
• Is Part Of: Chembiochem. Volume 14:Issue 14(2013)
• Page Start: 1745
• Page End: 1753
• Publication Date: 2013-09-05
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201300265
• Languages: English
• ISSNs: 1439-4227

Abstract

<abstract abstract-type="main" xml:lang="en"> <title>Abstract</title> <p>The Rieske/cytochrome <italic>b</italic> complexes, also known as cytochrome <italic>bc</italic> complexes, catalyze a unique oxidant‐induced reduction reaction at their quinol oxidase (Q<sub>o</sub>) sites, in which substrate hydroquinone reduces two distinct electron transfer chains, one through a series of high‐potential electron carriers, the second through low‐potential cytochrome <italic>b</italic>. This reaction is a critical step in energy storage by the Q‐cycle. The semiquinone intermediate in this reaction can reduce O<sub>2</sub> to produce deleterious superoxide. It is yet unknown how the enzyme controls this reaction, though numerous models have been proposed. In previous work, we trapped a Q‐cycle semiquinone anion intermediate, termed SQ<sub>o</sub>, in bacterial cytochrome <italic>bc</italic><sub>1</sub> by rapid freeze‐quenching. In this work, we apply pulsed‐EPR techniques to determine the location and properties of SQ<sub>o</sub> in the mitochondrial complex. In contrast to semiquinone intermediates in other enzymes, SQ<sub>o</sub> is not thermodynamically stabilized, and can even be destabilized with respect to solution. It is trapped in Q<sub>o</sub> at a site that is distinct from previously described inhibitor‐binding sites, yet sufficiently close to cytochrome <italic>b</italic><sub>L</sub> to allow rapid electron transfer. The binding site and EPR analyses show that SQ<sub>o</sub> is not stabilized by hydrogen bonds to proteins. The formation of SQ<sub>o</sub> involves "stripping" of both substrate ‐OH protons during the initial oxidation step, as well as conformational changes of the semiquinone and Q<sub>o</sub> proteins. The resulting charged radical is kinetically trapped, rather than thermodynamically stabilized (as in most enzymatic semiquinone species), conserving redox energy to drive electron transfer to cytochrome <italic>b</italic><sub>L</sub> while minimizing certain Q‐cycle bypass reactions, including oxidation of prereduced cytochrome <italic>b</italic> and reduction of O<sub>2</sub>.</p> </abstract>