Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Determination of the coupling relationships between the respiratory reactions and miscellaneous observations concerning rate-limiting steps.

The linear sequence of steps involved in the oxidation of extramitochondrial succinate by O2 in bovine heart mitochondria was examined by a steady-state kinetic method to determine whether or not freely diffusible intermediates occur between the various inhibitor-sensitive steps. The kinetic method is based on the facts (1) that if two inhibitor-sensitive steps within a sequence are linked by a freely diffusible intermediate, inhibition of one will make the other less rate limiting in the overall reaction and thus will increase the amount of inhibitor of the other step required for half-maximal inhibition of the overall reaction, and (2) that if the two steps are not linked in this manner, inhibition of one will make the other more rate limiting and thus will decrease the amount of inhibitor of the other required for half-maximal inhibition. These two types of "coupling relationships" between steps were designated as "sequential" and "fixed," respectively. The results indicate the existence of freely diffusible intermediates (sequential coupling relationships) between the succinate transport and succinate dehydrogenase reactions, between the succinate dehydrogenase and cytochrome bc1 reactions, and between the cytochromes bc1 and aa3 reactions. Uncoupling respiration from phosphorylation results in the coupling relationship between the bc1 and aa3 reactions becoming partially fixed. This change is accompanied by marked decreases in the degrees to which the bc1 and aa3 reactions limit the overall reaction and appears to account for the large uncoupler-induced releases of inhibition at the levels of the bc1 and aa3 reactions observed previously by others. It is suggested that cytochrome c is the freely diffusible intermediate between the bc1 and aa3 reactions and that the uncoupler-induced changes occur as a result of formation of functional and highly efficient supercomplexes between cytochrome c and the cytochromes bc1 and aa3 complexes.