Estimation of proton to adenosine 5'-triphosphate stoichiometry of Escherichia coli ATP synthase using phosphorus-31 NMR

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Abstract
High-field 31P NMR techniques were used to measure transmembrane .DELTA.pH in wild-type, unc A, and hem A mutants of E. coli. .DELTA..PSI. [transmembrane electrical potential] was measured by distribution methods with radioactive tetraphenylphosphonium bromide and 86Rb+ as the probes, while intracellular ATP, ADP Pi concentrations were determined from the 31P NMR spectra. .DELTA.G''P[phosphorylation potential] and the stoichiometry for ATP synthesis [.DELTA.G''p/(F.DELTA.p)] [F = Fanadays'' constant; .DELTA.p = protonmotive force] were then calculated. The stoichiometry of the ATP synthase was found to vary as a function of the cellular metabolic state. In nongrowing, wild-type cells .DELTA.p was 192 .+-. 16 mV with succinate as the substrate and saturating O2 tension. With limiting O2 (.simeq. 1 .mu.M O2), .DELTA.p was 125 .+-. 14 mV. Nucleoside triphosphate synthesis was observed in both cases. The H+/ATP stoichiometry varied from 2.15 .+-. 0.35 under aerobic conditions to 3.6 .+-. 0.8 at low O2 tension. .DELTA.p for unc A cells was 140 .+-. 14 mV with glucose as the substrate (> 2.5 .mu.M O2) and for hem A mutants was 115 .+-. 10 mV. The bulk phase potentials in O2-limited, wild-type cells and in respiratory deficient (hem A) cells are comparable, but in the former the ATPase is poised for synthesis while in the latter it generates .DELTA.p. The data support a role for localized interactions between the redox and the ATPase sites.