Quinol Oxidase Site Research Articles

A Functional Dimer Model of Ubiquinol Cytochrome C Oxidoreductase

Ubiquinol cytochrome c oxidoreductase (bc1 complex) serves as the electron junction in many respiratory systems. The enzyme possess both a quinol oxidase site (Qp-site) and a quinone reductase site (Qn-site) that operate together in a bifurcated electron transfer mechanism. While most electrons from complex I and II are funneled to cytochrome c, a small percent leak out of the respiratory system to produce the free radical, superoxide. In situ and other experimental systems, the enzyme exists as a dimer. But until recently, it was believed to operate as a functional monomer. Here we show that in order to explain the kinetic, superoxide, and antimycin stimulated data, a functional dimer model is required. A total of nine data sets consisting of kinetic and superoxide measurements from a range of experimental studies were used to constrain the models. The monomer and dimer models behave kinetically similar; however, the electron state distributions were vastly different. In the dimer catalytic mechanism, only one quinol oxidase site is active at any given time. Quinone reduction occurs randomly at either reductase site. But when antimycin A is bound to a single monomer, both Qp-sites are activated. With the superoxide production rate calibrated, model analysis reveals that the mammalian complex relative to the yeast and bacteria complexes either possess a less stable semiquinone at the Qp-site in or the estimated level of free radical scavenging is significantly underestimated.

Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

The deleterious effects of H2O2 on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H2O2 resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H2O2 on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H2O2 on respiratory complexes was almost completely recovered by β-mercaptoethanol treatment. H2O2 treatment resulted in full resistance to QO site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (QO) of complex III is the target for H2O2. H2O2 did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H2O2 addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.

Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex

The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The quinol oxidase (Q(o)) site in this complex oxidizes a hydroquinone (quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the "Rieske" iron-sulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a "Q-cycle" mechanism, which generates proton motive force for ATP synthesis. Since semiquinone intermediates of quinol oxidation are generally highly reactive, one of the key questions in this field is: how does the Q(o) site oxidize quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Q(o) site semiquinone (or quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O(2) reduction; 2) the Q(o) site semiquinone is highly stabilized making it unreactive toward oxygen; and 3) the Q(o) site catalyzes a quantum mechanically coupled two-electron/two-proton transfer without a semiquinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-cycle and superoxide production is essentially identical, consistent with model 1 but requiring modifications to models 2 and 3.

The Respiratory Substrate Rhodoquinol Induces Q-cycle Bypass Reactions in the Yeast Cytochrome bc1 Complex: MECHANISTIC AND PHYSIOLOGICAL IMPLICATIONS

The mitochondrial cytochrome bc(1) complex catalyzes the transfer of electrons from ubiquinol to cyt c while generating a proton motive force for ATP synthesis via the "Q-cycle" mechanism. Under certain conditions electron flow through the Q-cycle is blocked at the level of a reactive intermediate in the quinol oxidase site of the enzyme, resulting in "bypass reactions," some of which lead to superoxide production. Using analogs of the respiratory substrates ubiquinol-3 and rhodoquinol-3, we show that the relative rates of Q-cycle bypass reactions in the Saccharomyces cerevisiae cyt bc(1) complex are highly dependent by a factor of up to 100-fold on the properties of the substrate quinol. Our results suggest that the rate of Q-cycle bypass reactions is dependent on the steady state concentration of reactive intermediates produced at the quinol oxidase site of the enzyme. We conclude that normal operation of the Q-cycle requires a fairly narrow window of redox potentials with respect to the quinol substrate to allow normal turnover of the complex while preventing potentially damaging bypass reactions.

Proton release by the quinol oxidase site of the cytochrome b/c1 complex following single turnover flash excitation of intact cells of Rhodobacter capsulatus

(1) The myxothiazol-sensitive reduction of cytochrome (c, and c2) in anaerobic, intact cells of Rhodobacter capsulatus has a f1/2 of approx. 3 ms. The myxothiazol-sensitive component of the electrochromic absorbance change under similar conditions has ar1/2 of approx. 2.5 ms. It is concluded that the putative site for H+-release, the quinol oxidase (Qz) site of the cytochrome bc1 complex, turns over with t1/2 ≤ 3 ms. (2) Flash-induced H+-release from intact cells as measured with pH indicator is delayed: it is preceded by a short lag and has a t1/2 of approx. 28 ms. The extent and rate of H+-release are almost independent of the pH of the suspension between 6.0 and 8.0. (3) The average buffering capacity of the periplasmic and outer membrane region of Rb. capsulatus is 55 mM · pH−1 at pH 7.6. The effective diffusion coefficients for H+ and OH−1 in time-dependent relaxation phenomena are decreased by immobile buffering groups (Junge, W. and Polle, A. (1986) Biochim. Biophys. Acta 840, 265–273). A schematic model is proposed in whichH+ released at the outer face of the cytoplasmic membrane are delayed by a periplasmic region of homogeneous fixedbuffering groups. With reasonable assumptions about the thickness of the periplasmic region the model predicts the lag and the increased t1/2 for H+-release. (4) Based on independent measurements and assumptions the lower limit and upperlimit for the quantity of H+ released per quinol oxidised at the Qz. site of the cytochrome bc1 complex in intact cells are 1.41 and 3.14, respectively. A refinement of the data used to calculate the upper limit gives a more probable H+QH2 at Qz of 1.67. (5) The application of similar assumptions to estimate the quantity of charge translocated through the cytochrome bc1, complex per quinol oxidised at Qz yields values of 1.03, 1.22 and 1.06 for q/QH2 in accordance with the predictions of the protonmotive Q-cycle.

Role of the cytochrome b6.f complex in the redox-controlled activity of Acetabularia thylakoid protein kinase.

The regulation of the protein kinase activity responsible for the phosphorylation of the light-harvesting complex of photosystem II (LHCII) 27-kDa polypeptide involved in the State I-State II transitions in Acetabularia thylakoids was investigated. The LHCII kinase of isolated thylakoids retains its activity in absence of light-driven electron flow or reductants added in the dark. However, the kinase is reversibly inactivated by addition of oxidants in vitro or by far red (710 nm) light in vivo. Inhibitors of the quinol oxidase site of the cytochrome b6.f complex inactivate the LHCII kinase in the dark, and also in the light, or in presence of duroquinol when the plastoquinone pool is reduced. Inhibitors of the quinone reductase site of the b6.f complex have practically no effect in the dark and stimulate the kinase activity in the light. Based on these data and on our previous report, showing specific loss of LHCII kinase activity in a Lemna mutant lacking the cytochrome b6.f complex (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210), we propose that the activity of the LHCII kinase is regulated by the redox state of a cytochrome b6.f complex component(s) which responds to the balance of electron flow from photosystem II via the plastoquinone pool to photosystem I.

Inhibition of electron transfer and the electrogenic reaction in the cytochrome [formula omitted] complex by 2- n-nonyl-4-hydroxyquinoline N-oxide (NQNO) and 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB)

We investigated the interaction between the cytochrome b f complex and plastoquinone by determining the effect of the electron transport inhibitors 2- n-nonyl-4-hydroxy-quinoline N-oxide (NQNO) and 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB) on the flash-induced turnover of cytochromes b 6 and f, on the slow phase of the electrogenic reaction (515 s) and on steady-state electron transport. The experiments were performed using thylakoid membranes in the presence of duroquinol, methyl viologen, and diuron, conditions in which electron transport is driven by Photosystem I and includes the cytochrome b f complex. The data from these experiments indicate that NQNO and DBMIB inhibit at two different sites on the cytochrome b f complex. The data can be accommodated by a modified Q-cycle in which the primary site of inhibition by NQNO is the quinone reductase site (Q c) and the primary site of DBMIB inhibition is the quinol oxidase site (Q z). NQNO is envisioned to inhibit the oxidation of the cytochrome b 6-heme located nearest the outer aqueous phase, as well as to slow electron transfer between the two b-hemes. The effect of NQNO on the 515 s supports the notion that the slow electrogenic reaction is due to electron transfer between the two cytochrome b 6-hemes, followed by a reaction associated with plastoquinone reduction at the Q c-site (Jones and Whitmarsh (1985) Photobiochem. Photobiophys. 9, 119–127). The results indicate that the Q z-site and Q c-site are likely separated by 70% of the dielectrically weighted distance across the membrane. At low concentrations of DBMIB the kinetics of the 515 s and cytochrome b 6 reduction are monophasic, indicating that DBMIB moves from one inhibitory site to another within the turnover time of the cytochrome b f complex. This observation suggests that the debinding rate of DBMIB from the Q z-site is greater than 200 s −1. Lastly, comparing the effect of DBMIB on the extent of cytochrome f oxidation and the rate of cytochrome f reduction, raises the possibility that DBMIB has a second lower affinity binding site on the cytochrome b f complex, possibly interrupting electron flow between the FeS center and cytochrome f.

Reduction of cytochrome b-561 through the antimycin-sensitive site of the ubiquinol-cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides

Cytochrome b-561 of the ubiquinol-cytochrome c 2 oxidoreductase complex of Rhodopseudomonas sphaeroides is reduced after flash illumination in the presence of myxothiazol in an antimycin-sensitive reaction. Flash-induced reduction was observed over the redox range in which cytochrome b-561 and the Q-pool are both oxidized before the flash. The extent of reduction increased with increasing pH, and was maximal at pH > 10.0 where the extent approached that observed in the presence of antimycin following a group of flashes. Reduction of cytochrome b-561 in the presence of myxothiazol showed a lag of ~ 1 ms after the flash, followed by reduction with ( t 1 2 ~ 6 ms; by analogy with the similar kinetics of the quinol oxidase site, we suggest that the rate is determined by collision with the QH 2 produced in the pool on flash excitation.