The stoichiometry of charge translocation by cytochrome oxidase and the cytochrome bc₁ complex of mitochondria at high membrane potential

Abstract

The q '/2e stoichiometries (number of charges translocated per electron pair transferred) of cytochrome oxidase and the cytochrome bcl complex in rat liver mitochondria were determined at a range of membrane potentials up to 180 mV. The method used was similar to the one used in the preceding paper by us in this journal to determine the q+/O stoichiometry of the mitochondrial electron transport chain from succinate to oxygen. The measured 4+/2e stoichiometry of cytochrome oxidase was 3.5 positive charges per 0 atom reduced at low membrane potential (120 mV) and it decreased to about 1.5 at high membrane potential (180 mV). The measured qf/2e stoichiometry of the cytochrome bcl complex was between 1 and 1.25 positive charges ejected per electron pair and did not change significantly as Ay was varied from 85 mV to 157 mV. The sum of the qt/2e stoichiometries of cytochrome oxidase and the cytochrome bcl complex determined separately was similar to their value determined together for electron transport from succinate to oxygen over the range of membrane potentials studied. The most probable interpretation of these results is that the stoichiometry of the cytochrome bcl complex is invariant over a range of membrane potentials and that the q+/2e stoichiometry of cytochrome oxidase decreases from 4 at low membrane potential to 2 at high membrane potential.In the preceding paper 111 we showed that the q+/O stoichiometry for mitochondrial electron transport from succinate to oxygen decreases as Ay is increased (see also [2-51). This change in stoichiometry could occur either at the cytochrome bcl complex, at cytochrome oxidase or at both complexes.The q+/2e stoichiometry of the cytochrome bcl complex is generally agreed to be 2 at level flow, i.e. under conditions of vanishingly low Ap [6 -81. At intermediate values of Ay it does not appear to change [9,10]. The evidence at high values A y, is not as clear. Equilibrium thermodynamic measurements give the same value as at level flow [11, 121; however, there is evidence that the H+/2e stoichiometry of the mitochondrial cytochrome bcl complex incorporated into liposomes may be reduced from 4 to 2 at high (but not quantified) values of ApH [13]. Indirect evidence from the relationship between Av and the rate of charge efflux for mitochondria running only the cytochrome bcl complex has also been interpreted as showing slip in the complex [3].The 4+/2e stoichiometry of the cytochrome bcl complex of the photosynthetic bacterium Rhodobucter cupsulutus durCorrespondence to M. D. Brand, Department of Biochemistry, University of Cambridge, Cambridge, CB2 IQW, EnglandAbbreviations. H +/O, number of protons released to the external phase per oxygen atom reduced; .lo, rate of oxygen consumption; Ay, difference in electrical potential across the mitochondrial inner membrane; Ap, protonmotive force across the mitochondrial inner membrane; dpH, pH gradient across the mitochondrial inner membrane; 4+/2e, net number of positive charges translocated from the mitochondrial matrix to the exte...

David and Goliath: A Mitochondrial Coupling Problem?

“…Slip reactions in the mitochondrial respiratory complexes describe the mechanisms by which electrons can pass through a proton pump in the absence of proton movement, leading to a flexible H + /O ratio (Murphy and Brand, ). The occurrence of slipping processes is markedly dependent on temperature, being predominant at low temperatures, i.e., below 25°C (Canton et al., ), and negligible at 37°C (Brand et al., ; Canton et al., ).…”

“…This characteristic suggests that slip reactions might be of physiological importance in ectothermic animals, including common frogs in the present study. It is generally accepted that slip reactions involved in energy conversion are only evident for COX (Murphy and Brand, ; Papa et al., ). In addition, a causal relation seems to exist between the amount or activity of COX and the overall ATP synthesis yield (Groen et al., ; Nogueira et al., ).…”

Turkey cytochrome c oxidase contains subunit VIa of the liver type associated with low efficiency of energy transduction

“…Although it was generally assumed that cytochrome c oxidase from bacteria [15] and eukaryotes [16] has a constant H + /e − stoichiometry of 1.0, a decrease (‘slip’) down to H + /e − = 0.0 was described in cytochrome c oxidase, but not in the cytochrome bc1 complex, at high mitochondrial proton motive force Δp [17–21]. In addition, a variable ratio was measured with the reconstituted bovine heart enzyme, being 1.0 at low and 0.5 at high intraliposomal ATP/ADP ratios [22].…”

“…The H + /e − stoichiometry of cytochrome c oxidase is generally assumed to be 1.0 in bacteria [15], as well as in eukaryotes [16]. A ‘slip’ of proton pumping (decrease of H + /e − stoichiometry) at high proton motive force Δp, however, was repeatedly described [17–21]. Furthermore, subunit VIaH of the bovine heart enzyme was shown to induce a variable H + /e − stoichiometry, which is reduced from 1.0 to 0.5 at high intramitochondrial ATP/ADP ratios, with half‐maximal decrease at ATP/ADP = 100 [22].…”

Mitochondrial Calcium Signaling and Energy Metabolism

“…In both cases, influx of Ca 2+ through the Ca 2+ uniporter provides an inward current that depolarizes the mitochondria. Also during the depolarization, the activity of the respiration driven proton pumps increases due to less resistance because of the diminished membrane potential as suggested by Murphy and Brand 23. This is shown in Figure 3 D.…”