A semiquinone intermediate generated at the Q _o site of the cytochrome bc ₁ complex: Importance for the Q-cycle and superoxide production

Abstract

The cytochrome bc1 and related complexes are essential energyconserving components of mitochondrial and bacterial electron transport chains. They orchestrate a complex sequence of electron and proton transfer reactions resulting in the oxidation of quinol, the reduction of a mobile electron carrier, and the translocation of protons across the membrane to store energy in an electrochemical proton gradient. The enzyme can also catalyze substantial rates of superoxide production, with deleterious physiological consequences. Progress on understanding these processes has been hindered by the lack of observable enzymatic intermediates. We report the first direct detection of a semiquinone radical generated by the Q o site using continuous wave and pulsed EPR spectroscopy. The radical is a ubisemiquinone anion and is sensitive to both specific inhibitors and mutations within the Qo site as well as O2, suggesting that it is the elusive intermediate responsible for superoxide production. Paramagnetic interactions show that the new semiquinone species is buried in the protein, probably in or near the Qo site but not strongly interacting with the 2Fe2S cluster. The semiquinone is substoichiometric, even with conditions optimized for its accumulation, consistent with recently proposed models where the semiquinone is destabilized to limit superoxide production. The discovery of this intermediate provides a critical tool to directly probe the elusive chemistry that takes place within the Qo site.electron transfer ͉ free radical ͉ photosynthesis ͉ reactive oxygen species ͉ respiration T he cytochrome (cyt) bc 1 , b 6 f, and related complexes, collectively termed cyt bc complexes, are essential components of the respiratory and photosynthetic electron transport chains in mitochondria, many bacteria, and chloroplasts (1-3). These complexes oxidize quinol and reduce one-electron redox carriers while generating an electrochemical gradient of protons, termed the proton motive force (pmf ), which drives the synthesis of ATP and other bioenergetic processes. The natural substrate is ubiquinol (UQH 2 ) in the case of the mitochondrial and bacterial cyt bc 1 complexes, and the mobile carrier is cyt c in mitochondria or photosynthetic bacteria. The general mechanistic framework for the cyt bc complexes is the Q-cycle, first proposed by Mitchell (4-6) and modified by many others (e.g., refs. 7-14).In ''standard'' versions of the Q-cycle (2, 9, 15) a unique bifurcated oxidation of QH 2 occurs in the Q o site, located on the positively charged side (p-side) of the membrane. An initial single electron transfer to the ''Rieske'' 2Fe2S cluster produces a free radical semiquinone (SQ) intermediate (the anionic form or the neutral form, depending on the exact sequence of electron and proton transfers). The Rieske 2Fe2S cluster is the first in a series of carriers, termed the ''high potential chain,'' which in mitochondria and certain bacteria includes cyt c 1 followed by a soluble (or mobile) cyt c. Under normal conditions, the SQ intermediate ...

Movement of the Iron−Sulfur Head Domain of Cytochrome bc1 Transiently Opens the Catalytic Qo Site for Reaction with Oxygen

“…With those considerations, the experiments depicted in Figure 2 establish a broad range of conditions under which the sustained influx of electrons into heme b L cannot be balanced with the outflow of electrons from this heme to heme b H (Figure 1d,e). This traps heme b L in the reduced state which is expected to increase the probability of uncoupling of the FeS-and heme b L -mediated two-electron oxidation/reduction of quinol/ quinone at the Q o site and result in the formation of semiquinone (SQ o ) (22,31). The highly unstable SQ o is then expected to be able to reduce oxygen generating superoxide.…”

“…This, however, may change when the impeded electron flow outbalances reducing equivalents in the two chains, which has long been known from the classic experiments with an inhibitor antimycin which demonstrated that blocking specifically the electron flow through the Q i site is sufficient to make the enzyme vulnerable to superoxide production (10, 11). These kinds of observations have raised an ongoing debate about whether cytochrome bc 1 does produce superoxide in living cells (12)(13)(14)(15)(16) (5,(17)(18)(19)(20)(21)(22)(23)(24). Answering those questions not only would bring clarity to many physiological and medical studies but also should improve our understanding of the molecular mechanisms of energy conservation supported by cytochrome bc 1 .…”

Menaquinone as pool quinone in a purple bacterium

“…One might therefore expect that MKcontaining organisms simply ''take advantage'' of this additional driving force and get along with a single, UQ-type bc 1 complex. However, it has recently been shown that conservation of the Q-cycle driving force is critical to preventing deleterious superoxide production at the Q O site (32)(33)(34)(35). Substitution of the LP rhodoquinol (RQH 2 ) for UQH 2 in mitochondrial cyt.…”

Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release

“…The interaction between cytochrome c1 (cyto.c1) and cyto.c ensures the fast complex formation, optimal orientation and distance for electron transfer and also fast dissociation after electron transfer, which is critical for maintaining the electron flow and preventing the potential electron leak [4,5]. The cytochrome bc1 complex is also the major pathway for free radical generation within mitochondria [6][7][8]. Disruption of this interaction results in the loss of the oxidative phosphorylation, ample generation of reactive oxygen species (ROS) and disruption of mitochondrial functions and physiology leading to cell death.…”