Probing the Cl(-)-pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle.

Halorhodopsin (HR) functions as a light-driven inward Cl− pump. The Cl− transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl− was added to unphotolyzed Cl−-free NpHR–Brub complex, Brub caused the absorption spectral change in response to the Cl− binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl−-puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR′. These results indicate that Cl− is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR′ still maintains an altered protein conformation while another Cl− already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N–O quasi-equilibrium toward N, supporting Cl− release during the N to O transition. The membrane potential had a much larger effect on the Cl− transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl− transfers during N to O and O to NpHR′ transitions.