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 ...