Three-Step Isomerization of the Retinal Chromophore during the Anion Pumping Cycle of Halorhodopsin

The anion pumping cycle of halorhodopsin from Natronomonas pharaonis ( pHR) is initiated when the all- trans/15- anti isomer of retinal is photoisomerized into the 13- cis/15- anti configuration. A recent crystallographic study suggested that a reaction state with 13- cis/15- syn retinal occurred during the anion release process, i.e., after the N state with the 13- cis/15- anti retinal and before the O state with all- trans/15- anti retinal. In this study, we investigated the retinal isomeric composition in a long-living reaction state at various bromide ion concentrations. It was found that the 13- cis isomer (csHR'), in which the absorption spectrum was blue-shifted by ∼8 nm compared with that of the trans isomer (taHR), accumulated significantly when a cold suspension of pHR-rich claret membranes in 4 M NaBr was illuminated with continuous light. Analysis of flash-induced absorption changes suggested that the branching of the trans photocycle into the 13- cis isomer (csHR') occurs during the decay of an O-like state (O') with 13- cis/15- syn retinal; i.e., O' can decay to either csHR' or O with all- trans/15- anti retinal. The efficiency of the branching reaction was found to be dependent on the bromide ion concentration. At a very high bromide ion concentration, the anion pumping cycle is described by the scheme taHR -( hν) → K → L1a ↔ L1b ↔ N ↔ N' ↔ O' ↔ csHR' ↔ taHR. At a low bromide ion concentration, on the other hand, O' decays into taHR via O.