Identification of nucleus-encoded F0I protein of bovine heart mitochondrial H+-ATPase as a functional part of the F0 moiety

AbstractThe F0I protein of apparent Mr 27000, previously characterized [(1988) Eur. J. Biochem. 173, 1–8] as a genuine component of bovine heart F0, has been sequenced and shown to be identical with the nucleus encoded 24668 Da protein characterized earlier [(1987) J. Mol. Biol. 197, 89–100]. It is directly shown by proteolytic cleavage and reconstitution experiments that this protein, denoted here as PVP from the single-letter codes of the last three residues of the N-terminus, is involved in proton conduction by F0 and in its sensitivity to oligomycin

Mitochondrial F0F1 H+-ATP synthase Characterization of F0 components involved in H+ translocation

AbstractThe membrane F0, sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1-depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin-sensitive proton conduction, or recombined with purified F1 to give the oligomycin-sensitive F0F1-ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS-polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD-binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89–100], characterized as an intrinsic component of F0, (F0I, PVP protein [(1967) J. Biol. Chem. 242, 2547–2551]) is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin

The H(+)/e(−) stoicheiometry of respiration-linked proton translocation in the cytochrome system of mitochondria

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