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The H+-ATPase from chloroplasts was brought into the active, reduced state. Then, an electrochemica| potential difference of protons across the thylakoid membranes was F_,cnerated b~ ,in acid-base transition, ~lpH, combined with a K*/valinomycin diffusion potential. ~. The initial rate of ATP synthesis was measured with a rapid-mixing quenched-flow apparatus in the time-range between 20-150 ms. The rate of ATP symhesis depends in a sigmoidal way on ,dpH. Increasing diffusion potentials shifts the ApH-dcpendcncies to ~ower ~pH values. Analysis of the data indicate that the rate of ATP synthesss depends on the electrochemical potential diffcrenoe of protons irrespective of the relative contribution of ApH and Ch!oroplast; H~-ATPase; Enzyme-kinetics

Biophysical Chemistry / Chemical Kinetics / MembranesThe kinetics of proton transport coupled ATP synthesis at the chloroplast membrane was investigated upon energization of the membrane by an artificially generated ApH and an electric potential difference, Ay/. Using a rapid mixing system, rates of ATP synthesis were studied at short reaction times (< 150 ms) where all relevant parameters (A pH, A y/, substrate and product concentrations) remain practically constant at their initial values. Under these conditions it was found:1. The maximal rate of ATP synthesis obtained under these artificial conditions is the same as that obtained by light-induced ATP synthesis. 2. The turnover number of the ATPase is 410 s-'.3. The rate of ATP synthesis depends in a sigmoidal way on the transmembrane electrochemical potential difference of protons, A i H + , regardless of the relative contributions by A pH and A ty. 4.The functional dependence of the rate of ATP synthesis on A&+ does not reflect the kinetics of the catalytic events at the ATPase but the transformation of the ATPase from an inactive to a catalytically active state. 5 . The binding of two protons to the ATPase from the inside of the membrane is necessary for activation.

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