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.

Physiol Rev 95: 219 -243, 2015; doi:10.1152/physrev.00006.2014.-Mitochondrial respiration, an important bioenergetic process, relies on operation of four membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc 1 or ubiquinol: cytochrome c oxidoreductase), provides an electronic connection between these two diffusible redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme implicate that in addition to its well-defined function of contributing to generation of proton-motive force, cytochrome bc 1 may be a physiologically important point of regulation of electron flow acting as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic conditions. These features include the following: the opposing redox reactions at quinone catalytic sites located on the opposite sides of the membrane, the inter-monomer electronic connection that functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system, as well as the potential to generate superoxide and release it to the intermembrane space where it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding how cytochrome bc 1 may accomplish this regulatory physiological function, what is known and remains unknown about catalytic and side reactions within the quinone binding sites and electron transfers through the cofactor chains connecting those sites with the substrate redox pools. We also discuss the developed molecular mechanisms in the context of physiology of mitochondria.

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...

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.

During the operation of cytochrome bc1, a key enzyme of biological energy conversion, the iron−sulfur head domain of one of the subunits of the catalytic core undergoes a large-scale movement from the catalytic quinone oxidation Qo site to cytochrome c1. This changes a distance between the two iron−two sulfur (FeS) cluster and other cofactors of the redox chains. Although the role and the mechanism of this movement have been intensely studied, they both remain poorly understood, partly because the movement itself is not easily traceable experimentally. Here, we take advantage of magnetic interactions between the reduced FeS cluster and oxidized heme bL to use dipolar enhancement of phase relaxation of the FeS cluster as a spectroscopic parameter which with a unique clarity and specificity senses changes in the distance between those two cofactors. The dipolar relaxation curves measured by EPR at Q-band in a glass state of frozen solution (i.e., under the conditions trapping a dynamic distribution of FeS positions that existed in a liquid phase) of isolated cytochrome bc1 were compared with the curves calculated for the FeS cluster occupying distinct positions in various crystals of cytochrome bc1. This comparison revealed the existence of a broad distribution of the FeS positions in noninhibited cytochrome bc1 and demonstrated that the average equilibrium position is modifiable by inhibitors or mutations. To explain the results, we assume that changes in the equilibrium distribution of the FeS positions are the result of modifications of the orienting potential gradient in which the diffusion of the FeS head domain takes place. The measured changes in the phase relaxation enhancement provide the first direct experimental description of changes in the strength of dipolar coupling between the FeS cluster and heme bL.