Clarification of Factors Influencing the Nature and Magnitude of the Protonmotive Force in Bovine Heart Submitochondrial Particles

Abstract

The magnitude of the protonmotive force, and its division between pH gradient and membrane potential components has been further characterised in submitochondrial particles. In a reaction medium containing sucrose for osmotic support and 4-(2-hydroxyethyl)-l -piperazineethanesulfonate (Hepes) as buffer, with succinate as substrate, the total protonmotive force reached a maximum value of 245 mV. The presence of CI-enhanced the pH gradient with a partial but not fully compensating decrease in the membrane potential. When submitochondrial particles were suspended in a medium of low osmolarity consisting of phosphoric acid neutralised with Tris, again with succinate as substrate, the protonmotive force was lower and did not exceed 185 mV, and the pH gradient coniponent was equivalent to 25 mV or less.The final phosphorylation potential, AC,, maintained by the particles was higher in the phosphate/Tris medium (46-47.7 kJ mol-.') than in the sucrose/ Hepes/KCI medium (43.7 kJ rnol -'). Thus, comparison of the phosphorylation potential with the protonmotivc force would suggest that the mechanistic stoichiometry H'/ATP (H+ translocated per molecule of ATP synthesied) for the ATPase enzyme is 3 in the former medium and 2 in the latter, which might be taken to indicate two different types of mechanism required for ATP synthesis. However it is questioned whether a comparison of the protonmotive force with AC, in terms of equilibrium thermodynamics ought not to be complemented by analysis in terms of linear non-equilibrium thermodynamics. The latter treatment shows that it is possible to estimate only a value for the product of a phenomenological stoichiometry and the degree of coupling, which can be variable, but not the mechanistic stoichiometry. This treatment can also rationalise the observation of the higher AG, in reaction conditions where the lower values for Ap are estimated. Irrespective of possible explanations, the data show how an unprejudiced choice of reaction conditions can lead to different conclusions about the relationship between the phosphorylation potential and the protonmotive force.Although the acceptance of a chemiosmotic mechanism for membrane-energy transductions is widespread, the problem of quantitatively determining the thermodynamic capacity of the intermediate in chemiosmosis, the protonmotive force, remains.In previous experiments with submitochondrial particles we encountered the somewhat unexpected result that a higher protonmotive force was detected in a reaction mixture that contained sucrose, Hepes and KC1, compared with a reaction mixture that contained phosphoric acid neutralised with Tris base as the main ingredient [1,2]. The lower value for the protonmotive force found in the Pi/Tris medium was not paralleled by a lower value for the phosphorylation potential, so that on a thermodynamic basis a greater number of protons was apparently required per molecule of ATP synthesised in the P,/Tris medium [I], and also the lower protonmotive force could not be attributed to an uncouplingeffe...

THe proton-per-electron stoicheiometry of ‘site 1’ of oxidative phosphorylation at high protonmotive force is close to 1.5

“…As indicated by these authors, however, the AGP/AjiH+ ratios measured under conditions of ATP hydrolysis are expected to overestimate the actual stoicheiometry. Therefore the ratio found by Sorgato et al (1981) may still be consistent with an H+/ATP stoicheiometry of 2, as suggested by the experiments of Berry & Hinkle (1978), van Dam et al (1978) and Branca et al (1981) (the value of 1.9 in the medium of physiological osmosity is cited; it should be noted that in other media the latter authors find ratios exceeding the value of 2, or even 3).…”

“…It might be suggested that the endogenous proton permeability of the membranes of our submitochondrial particles was so high that already in the absence of added protonophore Aj + underestimated significantly AAH+' so that a ratio of AGe/AH+ =ne = 1 would be consistent with the observed ratio of AGe/ APH+= 1.6. Branca et al (1981) examined the dependence of the measured protonmotive force in succinate-energized submitochondrial particles on the medium composition. The medium composition used in the experiments reported in the present paper yielded the highest values of the protonmotive force, which would suggest that this medium minimizes the difference between A-H+ and AIH+.…”

Determination of the Proton Electrochemical Gradient across Biological Membranes

“…Similar conclusions have been drawn from double-inhibitor titration studies (Baum et al, 1971;Hitchens and Kell, 1982a;Westerhoff et al, 1982). Several reports contradict this (Padan and Rottenberg, 1973;Azzone et al, 1978c;Zoratti et al, 1981;Johnson and Hansford, 1977;Branca et al, 1981;Wilson and Forman, 1982;Sorgato et al, 1978;Holian and Wilson, 1980;see, however, Küster et al, 1981;Nicholls and Bernson, 1977;Nicholls, 1979), suggesting that ATP synthesis drains energy from the redox H + pump through a pathway which is not equivalent to Δ/Ι Η utilization for cation transport. The model proposes a stepwise activation of the ATPase depending on the number of H + ions interacting with the enzyme.…”

“…This discussion will be restricted to the stoichiometries in mitochondria (for reviews, see Krab, 1979, 1980;Brand, 1977;Papa, 1976;Mitchell, 1976;Boy er et al, 1977;Ferguson and Sorgato, 1982;Azzone et al, 1977b). Although most stoichiometric values have been obtained by measuring the ratio between input and output flow (flow ratio), it is also possible to calculate the stoichiometry of the single and coupled pumps from the ratio of input (X input ) and output (^output) f o r c e s (f or ce ratio) in state 4 (Rottenberg and Gutman, 1977;Branca et al, 1981;Lemasters and Billica, 1981). Although most stoichiometric values have been obtained by measuring the ratio between input and output flow (flow ratio), it is also possible to calculate the stoichiometry of the single and coupled pumps from the ratio of input (X input ) and output (^output) f o r c e s (f or ce ratio) in state 4 (Rottenberg and Gutman, 1977;Branca et al, 1981;Lemasters and Billica, 1981).…”