Primary photochemical events in halorhodopsin studied by subpicosecond time-resolved spectroscopy

Abstract

Primary photochemical events of the light-driven chloride-pump halorhodopsin (hR) are studied at room temperature by subpicosecond transient absorption measurements. On excitation of hR with a 600-nm, 0.6-ps pulse, excited-state absorption and stimulated emission appear immediately in the 420-530-nm and 650-770-nm wavelength regions, respectively, and both decay with a time constant of 2.3 ps. The calculated absorption spectrum of the excited state of hR (hR*) has a peak at 516 nm and a shoulder at about 460 nm. Accompanied by the decay of hR*, the primary ground-state product appears at around 645 nm. The quantum yield of the product formation is determined to be 0.27. The detailed analysis of the kinetics at 645 nm provided the faster rise time of the product (<1.0 ps) than the decay of the excited state (2.3 ps), as well as the possible presence of the J-intermediate (hRj). Instead of the simple sequential kinetic model considering three states of hR*, hRj, and hR, which has been applied to the primary process of bacteriorhodopsin, a parallel channel model is suggested for the primary process of hR. After Franck-Condon excitation, the cis-trans isomerization to hRj and the relaxation to hR* take place simultaneously. The latter decays only to hR via radiative and nonradiative processes. The present results suggest that the excited state having a reaction channel to cis-trans isomerization is not located at the potential minimum of hR* and that the relaxation process in the excited state is a process in competition with isomerization.

Bioenergetics of the Archaea

“…After absorption of light, the electronically excited state relaxes within the picosecond time scale to a redshifted photoproduct with maximal absorption at 600 nm (HR 600 ). The quantum yield of this reaction, about 0.3, is much lower than that determined for BR (268,400). The primary photoreaction involves an all-trans313-cis isomerization of retinal (127,154,202,303,399), and according to the FTIR difference spectrum between HR 600 and HR, this is quite similar to the K-BR difference spectrum (448).…”

Protein‐Controlled Ultrafast Photoisomerization in Rhodopsin and Bacteriorhodopsin

“…Essentially, I agree with this view, but I like to emphasize that the two -and three -state models are not suffi cient, because both models adopt only one coordinate on isomerization. Fluorescence spectroscopy of bovine Rh 40 and transient absorption spectroscopy of HR 70,71 clearly detected a prolonged excited state after photoisomerization was complete, which can be explained by neither two -nor three -state models. Another coordinate uncoupled to isomerization has to be taken into account, and personally I think that BR also possesses such a state.…”

“…14.9 ) after retinal photoexcitation was also reported by monitoring absorption of tryptophans. 73 Excited state is more long -lived in other archaeal -type Rhs, 70,71,74,75 and hence less effi cient for photoisomerization. These observations suggest that BR possesses the optimized structure for the primary photoisomerization mechanism.…”

Ultrafast Spectroscopy of Rhodopsins — Photochemistry at Its Best!

“…A comparison between bR and hR reveals that there is a correlation between ultrafast reaction rates and a high isomerization efficiency. The photoisomerization has a quantum yield of 0.6 63 for bR (J-formation time = 500 fs), but of only 0.3 29 for hR (f-formation time = 1000 fs). An interesting hypothesis is that a fast reaction rate results in a higher reaction quantum yield by cutting short the time during which the chromophore is present on the excited state.…”

“…The calculated curves (solid lines) represent an exponential fit to the data. While positive signals indicate increased absorption, negative signals indicate reduced absorption and/or stimulated emission (after ref29).…”