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Steady-state electron llow through and electron delivery into isolated dimeric bel complex (ubiquinol -cytochrome c oxidoreductase) from Neurospora crassa and beef heart mitochondria were studied in the presence of increasing concentrations of antimycin A, funiculosin and/or myxothiazol. Parabolic or linear inhibition curves were obtained, depending upon the different quinols and inhibitors that were used. Linear curves occur when the inhibitor directly affects the rate-determining step. The most reasonable explanation for the parabolic curves is given by a fast intradimeric exchange of the hydrophobic inhibitors antimycin A, funiculosin (rate < 500 s ~ I ) and of myxothiazol (rate > 1 s-') Using mitochondria from beef heart, the shape of the inhibition curve with antimycin A is parabolic if the quinol-02 oxidoreductase turns over at about 300 s-l, but hyperbolic if the rate is 5 times less. The hyperbolic titration curve may be the result of both intradimeric and an additional interdimeric redistribution (rate z 100 s -') of inhibitors between enzymes incorporated in a continuous phospholipid membrane. This explanation is supported by experiments with chromatophorcs obtained from Rhodobaeter capsulatus., cytochrome h becomes fully reoxidized within 1 s after a flash at substoichiometric entrations of antimycin A. This kinetic of the slow reoxidation can be expressed in terms of the intradimric and interdimeric redistribution with rate constants of about 10 s-' and 2 x lo6 M -l s -* , respectively. It seems that rapid inhibitor redistribution may be a widespread phenomenon for hydrophobic inhibitors of enzymes incorporated in lipid membranes.The mechanisms of redox-linked proton translocation by mitochondria1 bcl complex is now best described by the ubiquinone cycle proposed by Mitchell [l]. According to this mechanism, the enzyme contains two ubiquinone-catalytic centrcs, the QH2 (ubiquinol) oxidation centre o which is in contact with the positive aqueous phase and the ubiquinonereduction centre i which is in contact with the negative aqueous phase. For two QH2 molecules oxidized at centre 0, one ubiquinone molecule is reduced at the centre i, four protons are released into the intermembrane space, and two protons are taken up from the matrix space (for reviews see [2-41).Correspondence to G. Bechmann, Biophysics Division, University of Illinois, 156 Davenport Hall, 607 S. Mathews, Urhana, IIinois 61801, USA Abbreviations. DecQ, 2,3-dimethoxy-5-decyl-6-methyl-benzoquinone; DccQH,, 2,3-dimethoxy-5-decyl-6-methyl-benzoquinol; DQ, duroquinone, tetramethyl-benzoquinone; nQHzr duroquinol, tetramethyl-benzoquinol; MeQ, 2,3-dimethoxy-5,6-dimethyl-henzoquinone; MeQHz, 2,3-diniethoxy-5,6-dimethyl-benzoquinol; plastoquinol-0, 2,3-dimethyl-l,4-benzoquinol; uibquinol-2, 2,3-dimethoxy-5-methyl-6-geranyl-benzoquinol; ubiquinol-1 , 2,3-dimethoxy-5-methyl-6-isoprenyl-benzoquinol; 0, ubiquinone; QH,, ubiquinol; haem bH, high-potential haem of cytochrome b; SMPs, submitochondrial particles; MOA, (E)-p-methoxyacrylate.Enzyme. bcl...

Dimeric ubiquinol: cytochrome c reductase of Neurospora mitochondria was isolated as a protein-Triton complex and free of ubiquinol (Q). The enzyme was incorporated into phosphatidylcholine membranes together with Q. The effects of varying the molar ratio of Q to enzyme on the electron transfer from duroquinol (DHQ,) to the cytochromes c, c1 and b were studied.The rate of electron flow from DQH2 to cytochrome c was 15 times increased by Q and was maximal when one molecule of Q was bound to one enzyme dimer. The apparent K,,, value for DQH, of the Q-free enzyme was 5 pM and of the Q-supplemented enzyme 25 pM. The pre-steady-state rate of electron transfer from DQH, to cytochrome cI was also 15 times increased by Q and was maximal with one Q molecule bound to one enzyme dimer. This effect of Q was inhibited by antimycin. The pre-steady-state rate of electron transfer from DOH, to cytochrome b was 5 times decreased when Q was bound to the enzyme and this effect of Q was insensitive to myxothiazol. The Ht/2 e-stoichiometry with DQH, as substrate of the Q-supplemented enzyme was 3.6. These results are interpreted in accordance with a Q-cycle mechanism operating in a dimeric cytochrome reductase. Each enzyme monomer catalyses a single electron transfer from the QH,-oxidation centre to the Q-reduction centre and the two monomers cooperate in the reduction of Q to QH, at one Q-reduction centre. This centre contains two different binding sites for Q. DQH, does not properly react at the QH,-oxidation centre. DQH,, however, binds to the loose Q-binding site of the Q-reduction centre and reduces the Q bound to the tight Q-binding site of the centre. The QHz thus formed at the Q-reduction centre serves as electron donor for the QH,-oxidation centre.Cytochrome reductase (ubiquinol : ferricytochrome c oxidoreductase. EC 1.10.2.2) is one of the energy-transducing electron transfer complexes of the mitochondrial system of oxidative phosphorylation. The enzyme links the transfer of electrons from QH, to cytochrome c with the translocation of protons across the mitochondrial inner membrane (for reviews see [l -31). As a mechanism for this catalysis, the protonmotive Q-cycle proposed by Mitchell [4] is now widely discussed. According to the Q-cycle, the enzyme contains two Q-catalytic centres, the QH,-oxidation centre o which is located at the outer (protonically positive) side of the enzyme and the Q-reduction centre i located at the inner (protonically negative) side (see Fig. 11 in the Discussion). Successively, two protons are released from QH2 at centre 0, one electron is transferred via the iron-sulfur centre and cytochrome c1 to cytochrome c and the other electron via the low-potential and high-potential cytochromes b across the membrane to centre i. At centre i, a bound Q is first reduced to the radical anion Q-and after transfer of a second electron to centre i and association of two protons the reduction to QH2 is completed. New support for the Q-cycle mechanism has come from recent inhibitor studies which showed that ...

Ubiquinol:cytochrome‐c reductase, isolated from Neurospora mitochondria as a protein/Triton X‐100 preparation, and reconstituted into phospholipid membranes, catalyses the electron transfer from duroquinol to 2,3‐dimethoxy‐5‐decyl‐6‐methyl‐benzoquinone (decQ) on a myxothiazol‐insensitive, but antimycin‐sensitive, pingpong pathway. Duroquinol reacts first to form the altered, reduced enzyme E′. This reaction is followed by dissociation of duroquinone making way for E′ to bind decQ and convert it into decQH2.