Steady-state kinetics of the reduction of coenzyme Q analogs by complex I (NADH:Ubiquinone oxidoreductase) in bovine heart mitochondria and submitochondrial particles

The reduction kinetics of coenzyme Q (CoQ, ubiquinone) by NADH:ubiquinone oxidoreductase (complex I, EC 1.6.99.3) was investigated in bovine heart mitochondrial membranes using water-soluble homologs and analogs of the endogenous ubiquinone acceptor CoQ(10) [the lower homologs from CoQ(0) to CoQ(3), the 6-pentyl (PB) and 6-decyl (DB)analogs, and duroquinone]. By far the best substrates in bovine heart submitochondrial particles are CoQ(1) and PB. The kinetics of NADH-CoQ reductase was investigated in detail using CoQ(1) and PB as accepters. The kinetic pattern follows a ping-pong mechanism; the K-m for CoQ(1) is in the range of 20 mu M but is reversibly increased to 60 mu M by extraction of the endogenous CoQ(10). The increased K-m in CoQ(10)-depleted membranes indicates that endogenous ubiquinone not only does not exert significant product inhibition but rather is required for the appropriate structure of the acceptor site. The much lower V([)max] with CoQ(2) but not with DB as acceptor, associated with an almost identical K-m, suggests that the sites for endogenous ubiquinone bind 6-isoprenyl- and 6-alkylubiquinones with similar affinity, but the mode of electron transfer is less efficient with CoQ(2). The k(min) (k(cat)/K-m) for CoQ(1) is 4 orders of magnitude lower than the bimolecular collisional constant calculated from fluorescence quenching of membrane probes; moreover, the activation energy calculated from Arrhenius plots of k(min) is much higher than that of the collisional quenching constants. These observations strongly suggest that the interaction of the exogenous quinones with the enzyme is not diffusion-controlled. Contrary to other systems, in bovine submitochondrial particles, CoQ(1) usually appears to be able to support a rate approaching that of endogenous CoQ(10), as shown by application of the ''pool equation'' [Kroger, A., & Klingenberg, M. (1973) fur. J. Biochem. 39, 313-323] relating the rate of ubiquinone reduction to the rate of ubiquinol oxidation and the overall rate through the ubiquinone pool.