Release of respiratory control by uncouplers: The question of stoichiometry

Abstract

An alternative kinetic model of uncoupler action is envisaged in which the observed stoichiometry of uncoupler and titratable factor do not reflect chemical binding reactions. This model assumes (a) that highly effective uncouplers dissolve in the membrane, whereas less effective uncouplers remain largely in aqueous solution; (b) that a high affinity exists between respiratory chains and “low energy states”; (c) that maximal respiration requires the presence of only a small number of low-energy states and forms high-energy from low-energy states; (d) that uncouplers act by a bimolecular “collision” process without binding. In this model, respiratory chains must be capable of driving energy equivalents into a storage mode, with no effect on respiration rate until the store is almost filled. Results obtained with 2,4-dinitrophenol and S-13 show that the shapes of curves obtained when uncoupler concentration is plotted against respiration rate are the same for both “stoichiometric” and classical uncouplers. Mouse liver mitochondria, in the absence of added cations, showed a “titration” response to both dinitrophenol and S-13. Differences are: (1) the dinitrophenol titration point (approximately 35 μ m) is less dependent on mitochondrial concentration than that of S-13 (approximately 0.09 μ m) (for 1 mg of protein/ml); (2) a “lag” phase of some 30 sec occurs with S-13 at concentrations (approximately 0.1 μ m) giving the same uncoupling effect as 50 μ m-dinitrophenol (which shows no lag phase). Cytochrome b reduction level continues to fall upon DNP addition, and the rapid decline in per cent reduction of cytochrome b by a given increment of DNP ceases at approximately 35 μ m DNP, the concentration required for maximal respiration rate. Under some circumstances the release of respiration by uncoupler in the presence of azide shows an apparent titration independent of the final rate of respiration. This is true for DNP as well as for the apparent “stoichiometric” uncouplers. It is suggested that this does not necessarily identify a more specific uncoupling reaction than that observed in the absence of azide. The apparent release of azide inhibition by uucouplers is attributed to changes in the steady state level of cytochrome reduction on passing from state 3 to the uncoupled state, and to an oligomycin-like effect of azide on the mitochondrial ATPase.