DETECTING LOCALIZED PROTON CURRENTS IN PHOTOPHOSPHORYLATION BY PROCAINE INHIBITION OF THE TRANSTHYLAKOID PH-GRADIENT

The relationship between the transthylakoid pH-gradient, DELTApH, and the velocity of photophosphorylation, V(p), in thylakoid membranes from spinach was investigated using the local anesthetic amine procaine as inhibitor of DELTApH. When DELTApH was driven by Photosystem (PS) II + I-dependent electron flow, passing through the cytochrome b6/f complex, inhibition by procaine was accompanied by an increase of ATP formation. It appeared that procaine allowed for values of V(p) similar to those in controls (without procaine) at a significantly lower DELTApH than in the controls. In contrast, when DELTApH was driven by cyclic electron flow around PS I or by PS-II + I-dependent electron flow via a bypass around the cytochrome b6/f complex, or by PS II alone, procaine simultaneously caused an inhibition of DELTApH and a decrease of ATP formation. Inhibition of DELTApH by procaine did not induce an electrical membrane potential gradient that otherwise may have energetically compensated for the observed decline of DELTApH. The electron flow capacity was unaffected by procaine. However, inhibition of DELTApH did not significantly relax pH-dependent control of electron flux. Procaine accelerated ATP hydrolysis by pre-activated thylakoid ATPase to rates which were observed in the presence of uncouplers and had no direct effect on the activation state of the ATPase. The shift in the relationship between DELTApH and V(p) towards lower DELTApH persisted in thermodynamic equilibrium between the phosphorylation potential and DELTApH. The data indicated that the unconventional effect of procaine on photophosphorylation may be related to effects on proton translocation at the cytochrome b6/f complex and that a localized protonic coupling may occur between cytochrome b6/f and thylakoid-ATP-synthase complexes.