A Caged, Destabilized, Free Radical Intermediate in the Q‐Cycle

Abstract

The Rieske/cytochrome b complexes, also known as cytochrome bc complexes, catalyze a unique oxidant-induced reduction reaction at their quinol oxidase sites (Qo), 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 b. This reaction is a critical step in energy storage by the Q-cycle. The semiquinone intermediate in this reaction can reduce O2 to produce deleterious superoxide. It is yet unknown how the enzyme controls this reaction, though numerous models are proposed. In previous work we trapped 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 and properties of SQo in the mitochondrial complex. In contrast to semiquinone intermediates in other enzymes, SQo is not thermodynamically stabilized, and may even be destabilized with respect to solution. It is trapped in the Qo at a site, which is distinct from previously described inhibitor-binding sites, yet sufficiently close to cytochrome bL to allow rapid electron transfer. The binding site and EPR analysis show that SQo is not stabilized by hydrogen bonds to proteins. The formation of SQo involves “stripping” of both substrate -OH protons during the initial oxidation step, as well as conformational changes of the semiquinone and Qo 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 bL, while minimizing certain Q-cycle bypass reactions including oxidation of pre-reduced cytochrome b and reduction of O2.

The antimalarial compound ELQ‐400 is an unusual inhibitor of the bc1 complex, targeting both Qo and Qi sites

“…ISP and cytochrome c 1 ), SQ reactivity has been proposed to be limited by strongly destabilising this intermediate, lowering the probability of unwanted electron transfer bypass reactions. Here SQ is likely to be a mobile species within the Q o pocket without significant ligand–protein interactions during critical electron transfer reactions (although see for an alternative viewpoint).…”

Distinct properties of semiquinone species detected at the ubiquinol oxidation Q o site of cytochrome bc 1 and their mechanistic implications

“…[ 34 ] Vennam et al . [ 35 ] Sarewicz et al . [ 36 ] SQ o uncoupled SQ o –FeS spin-coupled transience of signal n.s.…”

The tetrahydrobiopterin radical interacting with high- and low-spin heme in neuronal nitric oxide synthase – A new indicator of the extent of NOS coupling

“…The pulsed EPR spectra were processed using the Xepr software package (Bruker-Biospin, Billerica, MA) or with custom MatLab (MathWorks) scripts. Analysis of coupled anisotropic spins follows the methods developed previously [40–42]. …”