Abstract
The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports SQ bound in the vicinity of reduced bH heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR-silent SQ bound close to oxidized bH, we calculated a midpoint redox potential (Em) of approximately 90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ.bH(3+) complexes. This favors recycling of the electron coming from center P and also implies a >2.5-fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre-steady-state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ.bH(3+) formation without inhibition of Q binding by QH2.
Highlights
The cytochrome bc1 complex recycles one of the two electrons from quinol (QH2) oxidation at center P by reducing quinone (Q) at center N to semiquinone (SQ), which is bound tightly
Bound at Center N—The purified yeast cytochrome bc1 complex has been reported to exhibit an EPR signal centered at g ϭ 2 attributed to an SQ radical [6]
We examined the properties of the EPR-detectable previously [32], bH reduction by DBH2 through center N showed biphasic kinetics when center P was inhibited with myxothiazol (Fig. 3A), whereas the presence of stigmatellin resulted in an additional re-oxidation phase that was abolished
Summary
The degree of reduction of the Rieske iron-sulfur cluster was followed by recording EPR spectra at 20 K (microwave frequency, 9.47 GHz; microwave power, 2 milliwatts; modulation amplitude, 0.64 milliteslas (mT); and sweep width, 100 mT). Showing normal titration behavior of the Rieske iron-sulfur cluster, no EPR signal in the range of SQ radicals was detectable in this sample. The reductive and oxidative titrations were averaged, and the resulting graph was fitted in the ORIGIN 5.0 program (OriginLab Corp.) to the following n ϭ 1 Nernst equation (Equation 1) with two components to obtain the redox potential for the bH (Em(bH)) and bL (Em(bL)) hemes as well as the relative contribution of the bH heme to the total absorbance (b),. The complete DynaFit script files are available as supplemental material
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