Complete tracking of transient proton flow through active chloroplast ATP synthase

Abstract

Proton pumping in thylakoid membranes and backflow of protons through the active ATP synthase CF0-CF1 (where CF0 is the proton channel and CF1 is the catalytic portion) were investigated by flash spectrophotometry. A steady pH difference across the membrane was generated by continuous measuring light, supplemented by voltage transients that were generated by flashing light. In the presence of P(i) and ADP, the electric potential transients elicited transients of proton flow via CF0-CF1, typically 1.3 H(+) per CF1 and flash group. Proton flow was blocked by CF0-CF1 inhibitors: N,N'-dicyclohexylcarbodiimide, acting on the channel component CF0, and tentoxin, acting on the catalytic component CF1. The half-rise time was 40 ms in (1)H(2)O and 78 ms in (2)H(2)O. ATP synthesis under conditions of flashing light and transient proton flow was characterized by a K(m)(P(i)) of only 14 muM, contrasting with a K(m) of several hundred micromolar for continuous ATP synthesis at high rate. This might reflect a resistance to P(i) diffusion. The degree of proton delocalization in the chemiosmotic coupling between redox reactions and ATP synthesis is under debate. In thylakoids, it has been proposed that intramembrane proton buffering domains act as ducts for protons between pumps and ATP synthases. In this work, transient proton flow by way of CF0-CF1 was completely tracked from the lumen, across the membrane, and into the suspending medium. Proton uptake from the lumen and charge flow across the membrane occurred synchronously and in stoichiometric proportion. The uptake of protons from the lumen by CF0-CF1, half completed in 40 ms, was preceded by release of protons from water oxidation into the lumen, half completed in <1 ms. Hence, pumps and ATP synthases were coupled through the lumen without involvement of intramembrane domains.