The H+/ATP ratios of H+-ATPsynthases from yeast mitochondria, spinach chloroplasts and E.coli

The H+/ATP ratio is an important parameter for the energy balance in cells as it defines the gearing between proton transport and proton coupled ATP synthesis. Based on the mechanism of rotational catalysis and the currently available structural information, it can be assumed that the H+/ATP ratio is equal to the ratio of c-subunits (H+ binding sites) to the number of β-subunits (ATP binding sites). The observed c-subunit numbers in different species imply the H+/ATP ratios be 3.3 for the mitochondrial ATPsynthase from yeast, 4.7 for the chloroplast ATPsynthase, and between 3.3 and 4.0 for the E.coli enzyme. We reconstituted the isolated F0F1s into liposomes and measured ATP synthesis or ATP hydrolysis as a function of pH at several constant stoichiometric products Q = [ATP]/([ADP][Pi ]). During reconstitution the internal phase of the liposomes was equilibrated with the acidic medium, so that the internal pH could be accurately measured with a glass electrode. An acid-base transition was carried out and the initial rate of ATP/ADP turnover measured for several pH at constant Q. The equilibrium pHeq for a given Q was obtained by interpolation, and each H+/ATP ratio was subsequently determined from the dependence of pHeq on the stoichiometric product Q.