(a) Molecular graphics model of rhodopsin illustrating the H-bond network extending across the retinal chromophore-binding pocket. Extracellular loop II (EII) is shown as the blue ribbon. The side chains of Ser-186 and Tyr-192 together with the backbone of Phe-91 and Cys-187 participating in the H-bond network are shown. (b) Glu-181 points toward the center of the polyene chain of retinal and the closest oxygen of Glu-181 is 4.7 Å away from C₁₂.

The pH dependence of the UV-visible absorption spectra of the WT rhodopsin (a–c), the E113Q mutant (d–f), and the E181Q mutant (g–i) before (blue) and after (red) photobleaching with light λ > 495 nm. The pigments were in 100 mM NaCl, 0.1% digitonin, and 0.5 Tris buffer at 4°C, a condition that traps native Meta I (λ_max = 480 nm). Two fully independent experiments produced identical spectral results.

UV-Vis spectra of the WT rhodopsin (a), and its S186A (b), S186I (c), and S186D (d) mutants purified in 0.1% DM in the dark (blue) and after illumination with light λ > 495 nm (red) at room temperature.

The photobleaching sequence of rhodopsin.

Preresonance Raman spectra of WT rhodopsin and its E181D and E181Q mutants. The spectra were taken by using preresonance excitation (30 mW, 765 nm) at a temperature of ≈0°C.

The pH dependence of UV-Vis absorption spectra of photobleached WT rhodopsin (Top), E113Q (Middle), and E181Q (Bottom) pigments. The pigments were studied in 100 mM NaCl, 0.1% DM at room temperature, a condition that stabilizes Meta II (λ_max = 380 nm) for the native pigment at neutral pH.

Schematic of the proposed proton transfer mechanism for switching the PSB counterion in rhodopsin. (a) Rhodopsin: two water molecules and Ser-186 form a H-bond chain between Glu-113 and Glu-181. Electrostatic interaction between the PSB and Glu-113 is indicated by the green dashed line (9). (b) Blue-shifted intermediate: after photoisomerization, the H-bond chain evolves so that the two water molecules and Ser-186 are lined up to prepare for the proton transfer and the PSB has shifted relative to Glu-113. (c) Lumirhodopsin: the PSB shifts further away from Glu-113 toward Glu-181. The gray arrows indicate a possible proton transfer pathway. Lumi is the transition state for the counterion switch as suggested by previous Raman and FTIR studies showing that the SB has moved from a strong H-bond environment to a very weak H-bond environment in the Lumi state (17–20). (d) Metarhodopsin I: proton transfer is completed. The PSB group is now close to Glu-181 to establish the electrostatic interaction (green dashed line) with the new counterion. In the Meta intermediates, it is possible that the counterion environment about Glu-181 is complex as has been observed and discussed in the case of bacteriorhodopsin (30, 31).