The Relative Proton Stoichiometries of the Mitochondrial Proton Pumps Are Independent of the Proton Motive Force

G C Brown

doi:10.1016/s0021-9258(18)63755-x

Abstract

Several different proton pumps were used to generate a proton motive force (delta p, proton motive force across the mitochondrial inner membrane) in isolated rat liver mitochondria, and the relationship between delta p and pump rate was investigated by titrating with various inhibitors of the pumps. It was found that this relationship was the same for mitochondria respiring on succinate irrespective of whether respiration was inhibited with malonate, antimycin or cyanide, indicating that the relationship was independent of the redox state of the respiratory chain. When delta p was generated by either the cytochrome bc1 complex, cytochrome oxidase, both together, or ATP hydrolysis (and transport), the reaction rates (in moles of electrons or ATP) were in the ratio of close to 3:1.5:1:1, respectively, at all accessible values of delta p. This suggests that the proton stoichiometries (H+/e and H+/ATP, where H+/e is the number of protons translocated vectorially across the inner membrane per electron transferred by the respiratory chain and H+/ATP is the number of protons translocated vectorially per ATP molecule hydrolyzed externally) were in the ratio of close to 1:2:3:3, respectively, at all values of delta p. Possible reasons for previous contradictory results are suggested.

Highlights

Several different protonpumps were used to gener- ently obtained when using different pumps [2, 3] or different ate aprotonmotiveforce (Ap, protonmotiveforce across the mitochondrial inner membrane) in isolated rat liver mitochondria, and the relationship between
They found that mitochondria respiring on glutamate respire at a rate proportional to the rate onsuccinate for all accessible values of Ap and concluded that the titrations could be accounted for by changes in the proton conductance without invoking slip
It. has been demonstrated many times [1,2,3,4,5,6,7,8,9] that when ionic conductance of the mitochondria and concluded that isolated mitochondria respiring on substrate in the absence there was both a substantial increase in the ionic conductance of phosphorylation are titrated with a respiratory inhibitor, of the mitochondria at high A# and a decrease in respiration can be substantially inhibited with only a small the q+/e (where $/eis the net number of positive charges drop inthe proton motiveforce motive forceacross the mitochondrial inner membra~e)T. wo stoichiometry of cytochrome oxidase, whereas the stoichiommain explanations have been put forward for this behavior: etry of the b c l complex wasinvariant

Summary

EXPERIMENTAL PROCEDURES

Substantially different inhibitor titration curves of Ap against Rat liver mitochondria were prepared as in [12]. Reaction rate (electron flux or ATP hydrolysis) were appar- ApH (where ApH is the difference in pH between the mitochondrial matrix and externaf phase) and A+ were measured as described in. €3.) and a research feflowship from St. Catharine’s College, Cam- centri ~ ~ nthge incubation to assay the distribution of isotopes bridge. The costs of publication of this article were defrayed in part between supernatant and pellet. Matrix volume was estimated from by the payment of page charges. This article must be hereby the difference in uptake into the pellet of 3Hz0 (5.0 gtCi/ml) and marked “advertisement” in accordance with 18 U.S.C. Section 1734 either [’4C]mannitol (0.2 pCi/ml) or [‘4C]sucrose (0.2 pci/mi), A$.

The Relative SPtrooitcohniometries of Mitochondria

RESULTS

Are the results reported here compatible with the relatively

DISCUSSION

The Relative Proton Stoichiometries of Mitochondria