The ubiquinol–cytochrome c oxidoreductases, central to cellular respiration and photosynthesis, are homodimers. High symmetry has frustrated resolution of whether cross-dimer interactions are functionally important. This has resulted in a proliferation of contradictory models. Here, we duplicated and fused cytochrome b subunits, and then broke symmetry by introducing independent mutations into each monomer. Electrons moved freely within and between monomers, crossing an electron-transfer bridge between two hemes in the core of the dimer. This revealed an H-shaped electron-transfer system that distributes electrons between four quinone oxidation-reduction terminals at the corners of the dimer within the millisecond time scale of enzymatic turnover. Free and unregulated distribution of electrons acts like a molecular-scale bus bar, a design often exploited in electronics.

In addition to its bioenergetic function of building up proton motive force, cytochrome bc1 can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQo) formed at the quinone oxidation/reduction Qo site (Qo) as a result of single-electron oxidation of quinol by the iron–sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme bL (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc1 that severely impeded the oxidation of FeS by cytochrome c1, changed density of FeS around Qo by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Qo and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQo that present a risk of generation of superoxide by cytochrome bc1. Isolation of this reaction sequence from multiplicity of possible reactions at Qo helps to better understand conditions under which complex III might contribute to ROS generation in vivo.

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...

This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome bc 1 and b 6 f (Cytbc 1 /b 6 f) membranous multisubunit homodimeric complexes. These remarkable molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes c or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient, which powers cellular metabolism in the majority of living organisms. Cytsbc 1 /b 6 f share many similarities but also have significant differences. While decades of research have provided extensive knowledge on these enzymes, several important aspects of their molecular mechanisms remain to be elucidated. We summarize a broad range of structural, mechanistic, and physiological aspects required for function of Cytbc 1 /b 6 f, combining textbook fundamentals with new intriguing concepts that have emerged from more recent studies. The discussion covers but is not limited to (i) mechanisms of energy-conserving bifurcation of electron pathway and energy-wasting superoxide generation at the quinol oxidation site, (ii) the mechanism by which semiquinone is stabilized at the quinone reduction site, (iii) interactions with substrates and specific inhibitors, (iv) intermonomer electron transfer and the role of a dimeric complex, and (v) higher levels of organization and regulation that involve Cytsbc 1 /b 6 f. In addressing these topics, we point out existing uncertainties and controversies, which, as suggested, will drive further research in this field.

One of the steps of a common pathway for biological energy conversion involves electron transfer between cytochrome c and cytochrome bc1. To clarify the mechanism of this reaction, we examined the structural association of those two proteins using the electron transfer-independent electron paramagnetic resonance (EPR) techniques. Drawing on the differences in the continuous wave EPR spectra and saturation recoveries of spin-labeled bacterial and mitochondrial cytochromes c recorded in the absence and presence of bacterial cytochrome bc1, we have exposed a time scale of dynamic equilibrium between the bound and the free state of cytochrome c at various ionic strengths. Our data show a successive decrease of the bound cytochrome c fraction as the ionic strength increases, with a limit of ∼120 mm NaCl above which essentially no bound cytochrome c can be detected by EPR. This limit does not apply to all of the interactions of cytochrome c with cytochrome bc1 because the cytochrome bc1 enzymatic activity remained high over a much wider range of ionic strengths. We concluded that EPR monitors just the tightly bound state of the association and that an averaged lifetime of this state decreases from over 100 μs at low ionic strength to less than 400 ns at an ionic strength above 120 mm. This suggests that at physiological ionic strength, the tightly bound complex on average lasts less than the time needed for a single electron exchange between hemes c and c1, indicating that productive electron transfer requires several collisions of the two molecules. This is consistent with an early idea of diffusion-coupled reactions that link the soluble electron carriers with the membranous complexes, which, we believe, provides a robust means of regulating electron flow through these complexes.