VOLTAGE-DEPENDENCE OF PROTON-PUMPING BY BACTERIORHODOPSIN IS REGULATED BY THE VOLTAGE-SENSITIVE RATIO OF M-1 TO M-2

The voltage dependence of light-induced proton pumping was studied wit h bacteriorhodopsin (bR) from Halobacterium salinarum, expressed in th e plasma membrane of oocytes from Xenopus laevis in the range -160 mV to +60 mV at different light intensities. Depending on the applied fie ld, the quenching effect by blue light, which bypasses the normal phot o and transport cycle, is drastically increased at inhibiting (negativ e) potentials, and is diminished at pump current increasing (positive) potentials. At any potential, two processes with different time const ants 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 i s 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 t he 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 Schif f base via the extracellular side. The results are supported by determ ination of photocurrents induced by bR on planar lipid films. The pH d ependence of the slow decay of M-1 is fully in agreement with the inte rpretation that the reprotonation of the Schiff base occurs from the e xtracellular side. The results give strong evidence that an externally applied electrical field changes the ratio of the M-1 and the M-2 int ermediate. As a consequence, the transport cycle branches into a nontr ansporting 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.