Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria.

The steady-state velocity dependence of the overall mitochondrial oxidative phosphorylation reaction on the concentrations of extramitochondrial ADP and P1 and of several of the catalytic components was investigated, using the O2 uptake step as the indicator reaction and conditions of saturation with O2, malate, and pyruvate. The studies were carried out with tightly coupled bovine heart mitochondria incubated in the presence of hexokinase, glucose, and Mg2+. The data were corrected to conditions of hexokinase saturation with factors determined in hexokinase dependence studies. The concentrations of catalytic components were varied, in effect, by application of highly specific, tight-binding inactivators of the components. The principal objectives were (a) to distinguish individual reactions coupled by freely diffusible intermediate reactants, (b) to determine the relationships (coupling relationships) between these reactions in regard to how a change in the degrees to which one limits the rate of the overall reaction affects the degree to which the others limit the rate, and (c) to use the findings to determine how the individual reactions are coupled. The feasibility of achieving these objectives was suggested by the observations (a) that the initial steady-state velocity of the overall reaction varies in fairly close accord with a rectangular hyperbola (i.e., with Michaelis-Menten kinetics) whether it is a catalytic component or a substrate that is varied, (b) that apparent Michaelis constants of the substrates and catalytic components may be used as indicators of the coupling relationships between the individual reactions, and (c) that two types of coupling relationships exist between the individual reactions: “sequential” (characteristic of reactions linked in simple sequence) and “nonsequential” (mechanism uncertain), in which a change in the degree to which one individual reaction of a pair is rate limiting results in an inverse change and in no change, respectively, in the degree to which the other is rate limiting. Six individual reactions were distinguished: the energy-yielding rotenone-, antimycin-, and cyanide-sensitive steps of the respiratory chain and the energy-consuming Pi transport, phosphorylation, and AdN (adenine nucleotide) transport reactions. The results indicate (a) that the coupling relationship is sequential between the Pi transport and rotenone-sensitive reactions, the Pi transport and cyanide-sensitive reactions, the AdN transport and rotenone-sensitive reactions, the AdN transport and cyanide-sensitive reactions, and the AdN transport and phosphorylation reactions, and (b) that the coupling relationship is nonsequential between the AdN and Pi transport reactions, the Pi transport and phosphorylation reactions, the Pi transport and antimycin-sensitive reactions, and the AdN transport and antimycin-sensitive reactions. In the sequential group of individual reaction pairs, the individual reactions of all but the AdN transport-phosphorylation reaction pair appear to be linked in a partially nonsequential manner. It is proposed that the nonsequential and partially nonsequential coupling relationships come about as a result of one individual reaction of a pair removing freely diffusible intermediate reactants at two or more points which are situated symmetrically and unsymmetrically, respectively, about the other.