Substrate redox potential controls superoxide production kinetics in the cytochrome bc complex.

The cytochrome (cyt1) bc1 (also known as Complex III or ubiquinol:cyt c oxidoreductase) and related complexes are essential energy transduction components in the respiratory and photosynthetic electron transport chains of a wide range of organisms (1–4). Its physiological role is to pump protons across the inner mitochondrial membrane to drive synthesis of ATP, but its detailed enzyme mechanism and possible roles in oxidative stress are still disputed. Our focus is on the mitochondrial cyt bc1 complex from Saccharomyces cerevisiae whose dimeric functional core is illustrated in cartoon form in the left hand side of Fig. 1. Each monomer consists of three essential catalytic subunits (2, 5–8): cyt b; the Rieske iron-sulfur protein (ISP); and cyt c1. The redox-active cofactors form two distinct redox chains leading away from the quinol oxidase (Qo) site on the positive side (p-side) of the membrane (5). A ‘low-potential chain’ extends across the membrane from the Qo site, through the cyt b subunit with its two b-type hemes (cyt bL and cyt bH), to the quinone reductase (Qi) site on the negatively-charged side (n-side) of the membrane. The ‘high-potential chain’ consists of the Rieske FeS cluster of the ISP, and the c-type heme of cyt c1. This chain, on the p-side of the membrane, relays electrons from the Qo site to a soluble cyt c. Figure 1 The uninhibited Q-cycle and superoxide production under partially inhibited conditions The cyt bc1 complex catalyzes a Q-cycle, illustrated in Fig. 1 Left, as first proposed by Mitchell and modified extensively (9–12) as a result of the work of many researchers. In most models of the Q-cycle (1, 13), QH2—originating from complex II in S. cerevisiae—binds in the Qo site and is oxidized in the so-called ‘bifurcated’ reaction, releasing two protons on the positively charged, p-side, of the membrane. The first QH2 electron enters the high-potential chain at the Rieske FeS cluster. A pivoting motion of the Rieske ISP (shown as two superimposed conformations in Fig. 1 Left) carries its FeS cluster close to cyt c1, allowing reduction of the cyt c1, which then relays the electron to a soluble cyt c. Eventually the electrons arrive at complex IV where they reduce O2 to water. In this model of the Q-cycle, the one-electron oxidation of QH2 at Qo forms a transient semiquinone (SQ) intermediate, termed SQo (14, 15). Instead of reducing the thermodynamically-favored high-potential chain a second time, SQo reduces cyt bL in the low-potential chain. Electrons sent into the low-potential chain travel through cyt bH and cyt bL, and eventually reduce a Q bound at the Qi site. After two turnovers of the Qo site, two electrons pass through the low potential chain, reducing Q to QH2 at the Qi site, with uptake of two protons from the n-side of the membrane. Overall, the Q-cycle oxidizes two QH2 at Qo, with the release of four H+ on the p-side; and reduces one Q at Qi with uptake of two H+ from the n-side. The net result is a highly efficient proton pump that helps produce the transmembrane proton motive force (pmf) that drives ATP synthase. This complicated Q-cycle gives a stoichiometry of 2 H+ translocated across the membrane per e− passed ultimately to cyt c in the steady state, increasing the ATP synthesized per O2 reduced (the P/O ratio) over that of a linear electron flow mechanism in which both QH2 electrons enter the high-potential chain (16, 17). It is argued that the bifurcated electron flow also serves to ‘quench’ the SQo (18–27) and prevent its reaction with O2 to generate superoxide. Superoxide production by the Qo site (and other reactions that ‘bypass’ the Q-cycle) is readily observed by blocking the low-potential chain in a number of ways, including: high pmf (28–30); high membrane potential (30); mutation of cyt b (17, 31); inhibition of the Qi site, e.g. by antimycin A (‘AA’ in Fig 1 Right); or inhibition of the Qo site niche near heme bL (proximal niche), e.g. by myxothiazole or methoxyacrylate (MOA) stilbene (‘X’ in Fig. 1 Right) (21, 22). Superoxide production at the Qo site is hotly debated as a mitochondrial source of superoxide and other reactive oxygen species in processes such as cell signaling, aging, carcinogenesis and metabolic diseases. A number of reactions ‘bypass’ the normal Q-cycle, reducing the efficiency of proton pumping and diverting electron flow, at least temporarily (20). The different bypass reactions are not mutually exclusive and might occur in combinations depending on conditions. We focus in this paper on the bypass reaction that produces superoxide during oxidation of quinol at the Qo site because of its very intimate relation to the initial steps in the Q-cycle. Recently, superoxide production has recently been described through a very different bypass reaction that involves reduction of quinone at the Qo site (32, 33), but it will not be considered here. Forquer et al. (34) recently showed that the rate limiting step for superoxide production in AA-inhibited mitochondrial cyt bc1 complex is the same as for the Q-cycle—involving reduction of the Rieske FeS cluster. Thus, the Q-cycle and superoxide production share intermediates along the same, or similar, reaction pathway(s). EPR of freeze-quenched bacterial cyt bc1 complex suggests that this common intermediate is SQo (14, 15). These recent results allow earlier conclusions about the order of reactions in the Q-cycle the bacterial Rhodobacter sphaeroides cyt bc1 complex (35) to be extended to the mitochondrial cyt bc1 complex. Little superoxide is generated by cyt bc1 under normal physiological conditions. Several classes of models attempt to explain how the Qo site steers the reaction along the Q-cycle and away from superoxide production (14, 15, 34, 35). The experimental evidence is increasingly at odds with models in which the oxidation of QH2 to SQ is radically different for superoxide production than for the Q-cycle, particularly the ‘double concerted’ electron transfer mechanisms (13, 25, 27, 36). Models that invoke conformational gating (6, 37–41) of the Qo site to steer electron flow are severely constrained because the same activating reaction occurs during quinol oxidation both when the putative gate functions ‘normally’ and when it leaks, to produce superoxide (34). The Q-cycle and the bypass reaction pathways must diverge at some point. On this point conflicting results have been obtained depending on reaction conditions. In contrast to Forquer et al. (34), Covian and Trumpower (42) concluded that there are different rate limiting steps for the Q-cycle and the bypass reactions from different activation energies under pre-steady state, non-substrate-saturated conditions. The relevance of these results to the reactions of the cyt bc1 complex is limited until the new rate limiting steps are identified and until it is determined which bypass reactions occur under these conditions. Consequently, we will restrict our discussion to substrate-saturated steady-state conditions unless explicitly stated. Important questions still remain concerning the sequence of reactions and the redox partner of the ISP in the rate limiting step. Does quinol transfer an electron directly to the FeS cluster thereby generating SQo or is there an intervening step? What factors control the rate of superoxide production with other quinols as substrate? Do interactions of the enzyme with particular sidegroups of the substrate guide the reaction and determine the products? To resolve these questions we examine superoxide production in the yeast mitochondrial cyt bc1 complex with a series of seven ubiquinol analogs designed to vary redox potentials and sidegroups on the quinol ring. One of the challenges to such work has been the limited ability to make consistent redox measurements on quinone species with the long, hydrophobic tails needed for activity. The ability to calculate these properties (43) now makes such a study feasible. We report here on the substrate properties that control the substrate saturated rate of superoxide production; we identify the rate limiting reaction; and we determine the sequence of the bifurcated electron transfer reactions.