H+/ATP coupling ratio at the unmodulated CF0CF1-ATP synthase determined by proton flux measurements

The H+/ATP coupling ratio is determined kinetically by comparing ATP synthesis and proton flow across the ATP synthase in spinach thylakoids under conditions of continuous illumination. Net proton flow at steady-state is zero, therefore the initial efflux in the dark is taken. A new method of kinetic analysis of the relaxation of the transmembrane proton gradient is presented, allowing direct determination of phosphorylating and basal proton fluxes at the same time. Special care is taken for the influence of dark phosphorylation and the H+ diffusion potential. The investigations give evidence of a H+/ATP coupling ratio of four for the oxidized or unmodulated state of the ATP synthase being at par with the coupling ratio for the reduced or modulated state of the enzyme.