Voltage dependence of proton pumping by bacteriorhodopsin is regulated by the voltage-sensitive ratio of M1 to M2

The voltage dependence of light-induced proton pumping was studied with bacteriorhodopsin (bR) from Halobacterium salinarum, expressed in the plasma membrane of oocytes from Xenopus laevis in the range -160 mV to +60 mV at different light intensities. Depending on the applied field, the quenching effect by blue light, which bypasses the normal photo and transport cycle, is drastically increased at inhibiting (negative) potentials, and is diminished at pump current increasing (positive) potentials. At any potential, two processes with different time constants for the M --> bR decay of similar to 5 ms (tau(1)) and similar to 20 ms (tau(2)) are obtained. At pump-inhibiting potentials, a third, long-lasting process with tau(3) approximate to 300 ms at neutral pH is observed. The fast processes (tau(1),tau(2)) can be assigned to the decay of M-2 in the normal pump cycle, i.e., to the reprotonation of the Schiff base via the cytoplasmic side, whereas tau(3) is due to the decay of M-1 without net pumping, i.e., the reprotonation of the Schiff base via the extracellular side. The results are supported by determination of photocurrents induced by bR on planar lipid films. The pH dependence of the slow decay of M-1 is fully in agreement with the interpretation that the reprotonation of the Schiff base occurs from the extracellular side. The results give strong evidence that an externally applied electrical field changes the ratio of the M-1 and the M-2 intermediate. As a consequence, the transport cycle branches into a nontransporting cycle at negative potentials. This interpretation explains the current-voltage behavior of bR on a new basis, but agrees with the isomerisation, switch, transfer model for vectorial transport.