Three-Step Isomerization of the Retinal Chromophore during the Anion Pumping Cycle of Halorhodopsin
2018
Kouyama, Tsutomu | Ihara, Kunio | Maki, Kosuke | Chan, Siu Kit
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.
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