Characterisation of Membrane Vesicles from Paracoccus denitrificans and Measurements of the Effect of Partial Uncoupling on Their Thermodynamics of Oxidative Phosphorylation

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Abstract
Vesicles from P. denitrificans were prepared by applying an osmotic shock to spheroplasts derived from cells that were grown anaerobically with succinate as C source and nitrate as electron acceptor. In the presence of phenazinemethosulfate or N,N,N''N'',-tetramethyl-p-phenylenediamine, the oxidation of isoascorbate supported the uptake of S14CN- and 86Rb+ (in the presence of valinomycin), whereas NADH and succinate oxidation resulted only in S14CN- uptake. The preparations contain right-side-out and inside-out vesicles and are related to the earlier proposal that the stimulation of an NADH-2,6-dichloroindophenol reductase activity by bee venom is an indicator of the proportion of right-side-out vesicles present. The implications impinge on previous conclusions about the mechanisms of sulfate and phosphate transport in P. denitrificans. The relationship between the protonmotive force (.DELTA.p; transmembrane proton electrochemical gradient expressed in mV) and the phosphorylation potential (.DELTA.Gp) generated by vesicles from P. denitrificans was studied as a function of the concentration of an uncoupler of oxidative phosphorylation. With NADH or succinate as substrate, the uncoupler had a more pronounced effect on .DELTA.p than on .DELTA.Gp, so that the ratio .DELTA.Gp/F .cntdot. .DELTA.p increased within a limited range of values of .DELTA.p close to the maximum. .DELTA.Gp/F .cntdot. .DELTA.p was approximately constant over the remaining range of .DELTA.p that was titrated. A fraction of highly coupled vesicles, separated from the initial preparation by centrifugation through a Ficoll pad, showed similar titration behavior. This demonstrated that heterogeneity within a vesicle preparation was not responsible for significant distortion of the true relationship between .DELTA.p and .DELTA.Gp. Values of .DELTA.p and .DELTA.Gp/F .cntdot. .DELTA.p (H+/ATP) from 143-108 mV and 3.9-4.4, respectively, were determined when NADH was substrate, whereas with succinate, .DELTA.p ranged from 123 to 88 mV and .DELTA.Gp/F .cntdot. .DELTA.p (H+/ATP) from 4.5 to 5.6. The variation in the value of .DELTA.GpF .cntdot. .DELTA.p, which can be equated with a minimum value for the H+/ATP of the ATP synthase enzyme, is similar to, but less pronounced than, some of the data previously reported for mitochondria. The observations with these bacterial vesicles, which represent an experimentally simpler system than mitochondria, might be taken as further evidence that measurements of the bulk phase .DELTA.p might not truly reflect the driving force for ATP synthesis sensed by the ATP synthase enzyme. Other explanations that would make the data consistent with a chemiosmotic mechanism cannot be eliminated. On a chemoiosmotic basis, and with recognition of possible errors in estimating .DELTA.p, it is concluded that the stoichiometry of proton translocation by the ATP synthase is probably 3 or 4, but not 2, for each ATP synthesized. An apparatus suitable for continuous detection of 14C-labeled solutes in the diffusate from a flow-dialysis cell is described.