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

Abstract

Cytochrome bc 1 , a key enzyme of biological energy conversion, generates or uses a proton motive force through the Q cycle that operates within the two chains of cofactors that embed two catalytic quinone oxidation/reduction sites, the Q o site and the Q i site. The Q o site relies on the joint action of two cofactors, the iron-sulfur (FeS) cluster and heme b L . Side reactions of the Q cycle involve a generation of superoxide which is commonly thought to be a product of an oxidation of a highly unstable semiquinone formed in the Q o site (SQ o ), but the overall mechanism of superoxide generation remains poorly understood. Here, we use selectively modified chains of cytochrome bc 1 to clearly isolate states linked with superoxide production. We show that this reaction takes place under severely impeded electron flow that traps heme b L in the reduced state and reflects a probability with which a single electron on SQ o is capable of reducing oxygen. SQ o gains this capability only when the FeS head domain, as a part of a catalytic cycle, transiently leaves the Q o site to communicate with the outermost cofactor, cytochrome c 1 . This increases the distance between the FeS cluster and the remaining portion of the Q o site, reducing the likelihood that the FeS cluster participates in an immediate removal of SQ o . In other states, the presence of both the FeS cluster and heme b L in the Q o site increases the probability of completion of short-circuit reactions which retain single electrons within the enzyme instead of releasing them on oxygen. We propose that in this way, cytochrome bc 1 under conditions of impeded electron flow employs the leak-proof short-circuits to minimize the unwanted single-electron reduction of oxygen.In respiratory and photosynthetic systems that couple electron transfer with a transmembrane proton gradient driving ATP production (1), cytochrome bc 1 (mitochondrial complex III) uses the Q cycle (2, 3) to catalyze electron transfer between quinone and cytochrome c. During the Q cycle, a reversible oxidation of quinol in the catalytic Q o site delivers one electron into the high-potential c-chain and the other into the low-potential b-chain. This reaction which is unique in biology relies on the energetic coupling of the two reduction/oxidation reactions, one involving the FeS 1 center of the c-chain and the other heme b L of the b-chain. The electrons are then exchanged between the FeS center and heme c 1 in the c-chain and among heme b L , heme b H , and the other quinone catalytic Q i site in the b-chain (Figure 1a) (3, 4). It appears that the two chains of cytochrome bc 1 have evolved to favor those productive electron transfers over the energy-wasting short-circuits of direct exchange of electrons between the chains or the uncontrolled leaks of electrons that produce damaging superoxide (5-9). Indeed, the enzyme working unperturbedly under driving force provided by substrates, quinol and cytochrome c, does not produce superoxide at detectable levels. This, however, may cha...

Electronic Connection Between the Quinone and CytochromecRedox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

“…This effect can be compared with the effect of the weak competitive inhibitor of the Q i site. As a result, at high range of UQH 2 concentrations, the activity of cytochrome bc 1 may decrease (29,35). This "downregulation" may play an important role in controlling the electron flux through the respiratory chain.…”

“…In reversibly operating Q o site, USQ o can be formed in two ways (29,82,145,189,209,220): as a part of forward reaction, when FeS withdraws electron from UQH 2 and the second electron transfer from USQ o to heme b L does not take place, or as a part of reverse reaction when heme b L donates an electron to UQ and the second electron transfer from FeS to USQ o does not take place. These two reactions can be referred to as "semiforward" and "semireverse," respectively (FIGURE 6).…”

“…In the semiforward scheme, reduced heme b L is unable to take electron from USQ o and thus cannot convert it to UQ (103,145,189,209). In the semireverse scheme, the reduced heme b L initiates a formation of USQ o (FIGURE 6) (29,82,220). As the semireverse reaction, unlike the semiforward reaction, uses UQ as a substrate, the recognition that the semireverse reaction may lead to ROS production came from analysis of a dependence of the rate of ROS production in submitochondrial particles and in isolated complexes on the redox state of the Q pool.…”

“…Further support for the semireverse scheme came from the analysis of various cofactor knockout mutants of cytochrome bc 1 (29). With these studies the model was further developed to describe an important constraint associated with the kinetic effects of the motion of the FeS head domain.…”

“…With these studies the model was further developed to describe an important constraint associated with the kinetic effects of the motion of the FeS head domain. The model proposes that probability of ROS generation increases as the distance, separating the FeS head domain from the Q o site at the exact time of USQ o formation, increases (29,220). This is because the positions of FeS remote from the Q o site diminish a chance that FeS engages in electron transfer to interact with USQ o (either by reducing USQ o to UQH 2 which would complete the reverse reaction, or oxidizing USQ o to UQ which would complete the short-circuit reaction) before USQ o reacts with oxygen.…”