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The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

ABSTRACT: A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active 'pulsed' state and the first one or two turnovers.

SUBMITTER: Papa S

PROVIDER: S-EPMC1152500 | biostudies-other | 1983 Nov

REPOSITORIES: biostudies-other

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Project description:1. The -->H(+)/e(-) quotients for proton release from mitochondria associated with electron flow from succinate and duroquinol to O(2), ferricyanide or ferricytochrome c, and from NNN'N'-tetramethyl-p-phenylenediamine+ascorbate to O(2), were determined from rate measurements of electron flow and proton translocation. 2. Care was taken to avoid, or to take into account, unrelated electron flow and proton translocation, which might take place in addition to the oxido-reductions that were the subject of our analysis. Spectrophotometric techniques were chosen to provide accurate measurement of the rate of consumption of oxidants and reductants. The rate of proton translocation was measured with fast pH meters with a precision of 10(-3) pH unit. 3. The -->H(+)/O quotient for succinate or duroquinol oxidation was, at neutral pH, 4, when computed on the basis of spectrophotometric determinations of the rate of O(2) consumption or duroquinol oxidation. Higher -->H(+)/O quotients for succinate oxidation, obtained from polarographic measurements of O(2) consumption, resulted from underestimation of the respiratory rate. 4. The -->H(+)/2e(-) quotient for electron flow from succinate and duroquinol to ferricyanide or ferricytochrome c ranged from 3.9 to 3.6. 5. Respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate by antimycin-inhibited mitochondria resulted in extra proton release in addition to that produced for oxidation of ascorbate to dehydroascorbate. Accurate spectrophotometric measurement of respiration showed that the -->H(+)/e(-) ratio was only 0.25 and not 0.7-1.0 as obtained with the inadequate polarographic assay of respiration. Proton release was practically suppressed when mitochondria were preincubated aerobically in the absence of antimycin. Furthermore, the rate of scalar proton consumption for water production was lower than that expected from the stoicheiometry. Thus the extra proton release observed during respiration elicited by NNN'N'-tetramethyl-p-phenylenediamine+ascorbate is caused by oxidation of endogenous hydrogenated reductants. 6. It is concluded that (i) the -->H(+)/O quotient for the cytochrome system is, at neutral pH, 4 and not 6 or 8 as reported by others; (ii) all the four protons are released during electron flow from quinol to cytochrome c; (iii) the oxidase transfers electrons from cytochrome c to protons from the matrix aqueous phase and does not pump protons from the matrix to the outer aqueous phase.

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The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

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