An attempt to discriminate catalytic and regulatory proton binding sites in membrane-bound, thiol-reduced chloroplast ATPase.
The question of the possible identity of catalytic and regulatory proton pathways in the chloroplast FoF1 ATPase has been studied using different energy-transfer inhibitors. Venturicidin, a reversible inhibitor of Fo, affects neither the delta mu H(+)-dependent thiol reduction of the membrane-bound chloroplast ATPase nor its ability to be activated by the proton gradient. It seems therefore to block only the proton flow required by the catalytic function of the enzymes. Venturicidin, however, also slows down the deactivation of the thiol-reduced ATPases during uncoupled ATP hydrolysis, following a delta mu H+ activation, but phloridzin, a reversible F1 inhibitor, has the same effect. Tentoxin, an irreversible F1 inhibitor, decreases the rate of ATP hydrolysis but does not affect the rate of deactivation. These findings suggest that catalytic and regulatory H(+)-binding sites are different. No distinction can be made, if any, between protons involved in unmasking the thiol-sensitive groups of F1 and in activating the enzyme. The effect of venturicidin and phloridzin on the deactivation is consistent with an inhibitory effect of newly formed--by ATP hydrolysis--ADP molecules, which might affect the enzyme without passing through the medium. Phosphate at millimolar concentration has an effect similar to low concentrations of phloridzin and venturicidin, probably by a simple back-reaction effect.