Steady-State Kinetics of the Reduction of Coenzyme Q Analogs by Complex I (NADH:Ubiquinone Oxidoreductase) in Bovine Heart Mitochondria and Submitochondrial Particles

Abstract

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 CoQ10 [the lower homologs from CoQ0 to CoQ3, the 6-pentyl (PB) and 6-decyl (DB) analogs, and duroquinone]. By far the best substrates in bovine heart submitochondrial particles are CoQ1 and PB. The kinetics of NADH-CoQ reductase was investigated in detail using CoQ1 and PB as acceptors. The kinetic pattern follows a ping-pong mechanism; the Km for CoQ1 is in the range of 20 microM but is reversibly increased to 60 microM by extraction of the endogenous CoQ10. The increased Km in CoQ10-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 Vmax with CoQ2 but not with DB as acceptor, associated with an almost identical Km, 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 CoQ2. The Kmin (kcat/Km) for CoQ1 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 kmin 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, CoQ1 usually appears to be able to support a rate approaching that of endogenous CoQ10, as shown by application of the "pool equation" [Kröger, A., & Klingenberg, M. (1973) Eur. 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.

Neurodegeneration in Methylmalonic Aciduria Involves Inhibition of Complex II and the Tricarboxylic Acid Cycle, and Synergistically Acting Excitotoxicity

“…Thus, the DBQ system is apparently more reliable to investigate complex II activity. Notably, it is accepted that DBQ resembles the natural ubichinone chemistry in mitochondria (30). Surprisingly, we found no evidence for an inhibition of complex II at concentrations of up to 20 mM MMA neither in the DBQ (0.5-20 mM: 107-111% of control) nor PMS systems (109 -115% of control; Table I).…”

Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation

“…Using HAR, the upper limit of NADH/Q activity of complex I was calculated to be 14 Ϯ 1 units/mg complex I. These values of complex I NADH/Q activity fall in the middle of estimates of complex I NADH oxidase activity in SMPs, which range from ϳ4 to 30 units/mg complex I measured using a variety of Q analogues (45,(52)(53)(54). Our value of 12-14 units/mg agrees closely with the NADH/DQ activity of ϳ14 units/mg complex I measured in bovine heart SMPs by Fato et al (originally reported as k cat ϭ 225 s Ϫ1 (52)).…”

NADH Oxidation by the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

“…The protein concentration of the flavin domain and the Fe-S domain was standardized by UV spectroscopy using the calculated extinction coefficients at 280 nm based on the content of aromatic amino acid residues of the apoproteins. The concentrations of NADH (⑀ 340 ϭ 6.22 mM (30) and FAD (⑀ 450 ϭ 11.3 mM Ϫ1 cm Ϫ1 ) (31) were calculated based on their absorption coefficients.…”