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The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential.

ABSTRACT: 1. The magnitude of the protonmotive force in respiring bovine heart submitochondrial particles was estimated. The membrane-potential component was determined from the uptake of S14CN-ions, and the pH-gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate the membrane potential was approx. 145mV and the pH gradient was between 0 and 0.5 unit when the particles were suspended in a Pi/Tris reaction medium. The addition of the permeant NO3-ion decreased the membrane potential with a corresponding increase in the pH gradient. In a medium containing 200mM-sucrose, 50mM-KCl and Hepes as buffer, the total protonmotive force was 185mV, comprising a membrane potential of 90mV and a pH gradient of 1.6 units. Thus the protonmotive force was slightly larger in the high-osmolarity medium. 3. The phosphorylation potential (= deltaG0' + RT ln[ATP]/[ADP][Pi]) was approx. 43.1 kJ/mol (10.3kcal/mol) in all the reaction media tested. Comparison of this value with the protonmotive force indicates that more than 2 and up to 3 protons must be moved across the membrane for each molecule of ATP synthesized by a chemiosmotic mechanism. 4. Succinate generated both a protonmotive force and a phosphorylation potential that were of similar magnitude to those observed with NADH as substrate. 5. Although oxidation of NADH supports a rate of ATP synthesis that is approximately twice that observed with succinate, respiration with either of these substrates generated a very similar protonmotive force. Thus there seemed to be no strict relation between the size of the protonmotive force and the phosphorylation rate. 6. In the presence of antimycin and/or 2-n-heptyl-4-hydroxyquinoline N-oxide, ascorbate oxidation with either NNN'N'-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine as electron mediator generated a membrane potential of approx. 90mV, but no pH gradient was detected, even in the presence of NO3-. These data are discussed with reference to the proposal that cytochrome oxidase contains a proton pump.

SUBMITTER: Sorgato MC

PROVIDER: S-EPMC1185904 | biostudies-other | 1978 Jul

REPOSITORIES: biostudies-other

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Project description:1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S(14)CN(-), and the transmembrane pH gradient component from the uptake of [(14)C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN'N'-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (DeltaG(p)=DeltaG(0)'+RTln[ATP]/ [ADP][P(i)]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO(3) the protonmotive force was not detectable (<60mV) but DeltaG(p) was not altered. This result may indicate either that there is no relationship between the protonmotive force and DeltaG(p), or that for an unidentified reason the equilibration of SCN(-) or methylamine with the membrane potential and the pH gradient is prevented by NO(3) (-) in this system.

Project description:In an attempt to establish the relative importance of diffusional and chemical control in the reactivity of the two of the two substrates, ubiquinol and cytochrome c, we have undertaken as extensive characterization of the steady-state kinetics of ubiquinol-cytochrome c reductase (EC 1.10.2.2) when present in open submitochondrial particles from bovine heart. The kinetic pattern follows a Ping Pong mechanism; contrary to the situation found with the isolated enzyme [Speck and Margoliash (1984) J. Biol. Chem. 259, 1064-1072, and confirmed in our laboratory], no substrate inhibition by oxidized cytochrome c was observed with the membrane-bound enzyme. Endogenous oxidized ubiquinone-10 is unable to exert product inhibition under the conditions employed. In the Ping Pong mechanism for this enzyme, the reaction scheme can be clearly divided into two parts, and the Kmin. (kcat./km) value for one substrate is independent of the rate constant for the second substrate. Both ubiquinol-1 and ubiquinol-2 can be used as electron donors reacting with the enzyme from within the lipid bilayer [Fato, Castelluccio, Palmer and Lenaz (1988) Biochim. Biophys. Acta 932, 216-222]; the kmin. values for ubiquinols, when calculated on the basis of their membranous concentrations, are significantly lower than the kmin. for cytochrome c. The temperature-dependence of the kinetic parameters was investigated by titrating each of the substrates under quasi-saturating concentrations of the second substrate. Arrhenius plots of Vmax. extrapolated from both cytochrome c and ubiquinol titrations were linear, when care was taken to verify the quasi-saturating concentrations of the fixed co-substrate. The Arrhenius plots for the kmin. values for both ubiquinol and cytochrome c were linear, but the activation energy was much higher for the former, particularly when calculated for ubiquinol dissolved in the lipid phase; the very low value of activation energy of the kmin. for cytochrome c is strong support for diffusion control being present in the reaction of cytochrome c with the membranous enzyme. In contrast to the soluble enzyme, ubiquinone titrations of submitochondrial particles at low cytochrome c concentrations deviated from hyperbolic behaviour. Changing the medium viscosity with either poly(ethylene glycol) or sucrose had a strong effect on the cytochrome c kmin., whereas the change in the ubiquinol kmin. was much smaller. From the viscosity studies the extent of diffusional control could be calculated, revealing that the reaction with cytochrome c was mostly diffusion-limited. The viscosity of the membrane was changed by incorporating cholesterol; no significant effect on the ubiquinol kmin. ascribable to diffusion control could be recognized.(ABSTRACT TRUNCATED AT 400 WORDS)

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The protonmotive force in bovine heart submitochondrial particles. Magnitude, sites of generation and comparison with the phosphorylation potential.

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