Nature of the chromophore binding site of bacteriorhodopsin: the potential role of Arg82 as a principal counterion.

The nature of the chromophore binding site of light-adapted bacteriorhodopsin is analyzed by using modified neglect of differential overlap with partial single and double configuration interaction (MNDO-PSDCI) molecular orbital theory to interpret previously reported linear and nonlinear optical spectroscopic measurements. We conclude that in the absence of divalent metal cations in close interaction with Asp85 and Asp212, a positively charged amino acid must be present in the same vicinity. We find that models in which Arg82 is pointed upward into the chromophore binding site and directly stabilizes Asp85 and Asp212 are successful in rationalizing the observed one-photon and two-photon properties. We conclude further that a water molecule is strongly hydrogen bonded to the chromophore imine proton. The chromophore "1Bu*+" and "1Ag*-" states, despite extensive mixing, exhibit significantly different configurational character. The lowest-lying "1Bu*+" state is dominated by single excitations, whereas the second-excited "1Ag*-" state is dominated by double excitations. We can rule out the possibility of a negatively charged binding site, because such a site would produce a lowest-lying "1Ag*-" state, which is contrary to experimental observation. The possibility that Arg82 migrates toward the extracellular surface during the photocycle is examined

Vertebrate ultraviolet visual pigments: Protonation of the retinylidene Schiff base and a counterion switch during photoactivation

For visual pigments, a covalent bond between the ligand (11-cis-retinal) and receptor (opsin) is crucial to spectral tuning and photoactivation. All photoreceptors have retinal bound via a Schiff base (SB) linkage, but only UV-sensitive cone pigments have this moiety unprotonated in the dark. We investigated the dynamics of mouse UV (MUV) photoactivation, focusing on SB protonation and the functional role of a highly conserved acidic residue (E108) in the third transmembrane helix. On illumination, wild-type MUV undergoes a series of conformational changes, batho → lumi → meta I, finally forming the active intermediate meta II. During the dark reactions, the SB becomes protonated transiently. In contrast, the MUV-E108Q mutant formed significantly less batho that did not decay through a protonated lumi. Rather, a transition to meta I occurred above ≈240 K, with a remarkable red shift (λ(max) ≈ 520 nm) accompanying SB protonation. The MUV-E108Q meta I → meta II transition appeared normal but the MUV-E108Q meta II decay to opsin and free retinal was dramatically delayed, resulting in increased transducin activation. These results suggest that there are two proton donors during the activation of UV pigments, the primary counterion E108 necessary for protonation of the SB during lumi formation and a second one necessary for protonation of meta I. Inactivation of meta II in SWS1 cone pigments is regulated by the primary counterion. Computational studies suggest that UV pigments adopt a switch to a more distant counterion, E176, during the lumi to meta I transition. The findings with MUV are in close analogy to rhodopsin and provides further support for the importance of the counterion switch in the photoactivation of both rod and cone visual pigments