Steady-state kinetics of high molecular weight (type-I) NADH dehydrogenase.
(1) Studies of the steady-state kinetics of the NADH dehydrogenase activity of Complex I (NADH: Q oxidoreductase) revealed that the reaction mechanism with the one-electron acceptor ferricyanide or the two-electron acceptor 2,6-dichloro-indophenol is ping pong bi bi, with double substrate inhibition. NADH inhibits the reaction of the reduced form of the flavoprotein with the acceptors, and the acceptors prevent NADH from reacting with the oxidized form. This implies that both NADH and acceptors react with the same site on NADH dehydrogenase. (2) The velocity at infinite NADH and acceptor concentrations (corrected for the double substrate inhibition) is much larger with ferricyanide than with the indophenol. It is concluded that the latter binds to the reduced enzyme. Thus, with ferricyanide the rate constant measured refers to the dissociation of bound NAD+ from the reduced enzyme (k2) and with the indophenol to the rate constant of oxidation of reduced enzyme by bound acceptor (k4). The latter value is not an estimate for the situation in vivo, where ubiquinone is the acceptor. (3) The rate constant of the dissociation of bound NAD+ from the reduced enzyme (k2) increases with pH. It is suggested that an ionizing group on the enzyme is involved in the dissociation. (4) After extraction of ubiquinone from Complex I with pentane curve relating activity at infinite ferricyanide concentration to NADH concentration changes from hyperbolic to sigmoidal. The hyperbolic curve is restored by reincorporating ubiquinone. It is concluded that ubiquinone is an effector for NADH dehydrogenase.
D015086:2,6-Dichloroindophenol / Q000494:pharmacology; D000818:Animals; D005292:Ferricyanides / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010084:Oxidation-Reduction
D005292:Ferricyanides; D007527:Isoenzymes; D009243:NAD; D015086:2,6-Dichloroindophenol; D009247:NADH, NADPH Oxidoreductases