Stoichiometric aspects of the uncoupling of oxidative phosphorylation by carbonyl cyanide phenylhydrazones

Abstract

Heavy beef heart mitochondria (HBHM) were titrated with carbonyl cyanide m-chlorophenylhydrazone and with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (pF 3 CO-CCP) under a variety of conditions. For 100% uncoupling as little as one molecule of uncoupler sufficed for as many as 27 potential coupling sites. From a determination of the maximal experimental respiratory rate of the HBHM it was possible to calculate the “concentration of active chains” in each experiment. Significantly, an almost exact stoichiometry was found between the uncoupler and the number of “active phosphorylation sites” (“active chains” × control P O ). The possible constancy of this stoichiometric relationship was further tested by studying the degree of uncoupling resulting from the addition of varying levels of uncouplers to mitochondria exhibiting varying initial P O ratios and respiring at rates that were varied by use of the respiratory inhibitors rotenone, malonate, and cyanide. All the data were consistent with the concept that the uncouplers act preferentially on “active phosphorylation sites,” and that this affinity relates to the existence of a high-energy intermediate rather than to the state of reduction of these sites. The “stoichiometric efficiency” (i.e., the degree of uncoupling per molecule of uncoupler per active phosphorylation site) was determined over a wide range of respiratory rates (maximal obtainable respiratory rate/observed rate in the range between 3 and 40 or more). Over this entire range there was only a marginal overall decline in the former parameter; over 80% of the observed points fell between 0.7 and 1.4. The maintenance of a constant stoichiometry supports the concept that uncouplers such as pF 3 CO-CCP are highly mobile within the mitochondrial membrane and have a very high affinity only for actively functioning coupling sites. The amount of uncoupler required to produce a given degree of uncoupling is usually dependent only upon the rate of generation of high-energy intermediates.