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Pre-steady-state reduction kinetics of QH2:cytochrome c oxidoreductase and the Q-pool: Evidence for a special quinone not in rapid equilibrium with the Q-pool

1987; Elsevier BV; Volume: 892; Issue: 1 Linguagem: Inglês

10.1016/0005-2728(87)90257-x

1879-2650

Alfred N. Van Hoek, M.C.M. van Gaalen, S. de Vries, J.A. Berden,

Coenzyme Q10 studies and effects

The pre-steady-state kinetics of the reduction of the prosthetic groups of QH2: cytochrome c oxidoreductase in bovine heart submitochondrial particles were studied in relation to the kinetics of the Q-10 reduction, using duroquinol as substrate. The prosthetic groups, including semiquinone, were measured with EPR and low-temperature-diffuse reflectance spectroscopy, the samples being prepared with the rapid-freeze quench technique. For the determination of the redox state of ubiquinone in the pre-steady state the rapid chemical quench technique was used as an extension of the rapid-freeze quench technique, and Q-10 and QH2-10 were measured with reversed-phase HPLC after extraction with petroleum ether. Ubiquinone was reduced biphasically, 8% of total Q-10 (equal to 1 mol Q-10/mol cytochrome c1, being reduced within 5 ms, and the rest, the Q-pool, at a much lower rate. The initial rapid reduction of this special Q-10 was accompanied by rapid formation of Q⨪i and rapid reduction of a large part of the cytochrome b-562. Both semiquinone formation and reduction of b-562 showed transient kinetics due to a contribution of the reaction pathway via centre o when the iron-sulphur cluster and cytochrome c1 were oxidised. The majority of the special quinol was located at centre i, probably bound, but also at centre o some bound quinol was formed. This was visible when antimycin was present, the antimycin-insensitive bound quinol being totally sensitive to myxothiazol. Myxothiazol alone accelerated the reduction of the Q-pool via centre i, but also the equilibration of cytochrome b-562 with the Q-pool. Antimycin drastically lowered the rate of reduction of the Q-pool and additionally seemed to block the rapid electron transfer from part of the Rieske iron-sulphur cluster to cytochrome c1. It is concluded that, during the pre-steady-state, cytochrome b-562 is not in equilibrium with the Q-pool and that the rate of equilibration is probably determined by the rate of dissociation of the special bound quinol from centre i.