The nature of the primary photochemical events in rhodopsin and isorhodopsin.

The nature of the primary photochemical events in rhodopsin and isorhodopsin is studied by using low temperature actinometry, low temperature absorption spectroscopy, and intermediate neglect of differential overlap including partial single and double configuration interaction (INDO-PSDCI) molecular orbital theory. The principal goal is a better understanding of how the protein binding site influences the energetic, photochemical, and spectroscopic properties of the bound chromophore. Absolute quantum yields for the isorhodopsin (I) to bathorhodopsin (B) phototransformation are assigned at 77 K by using the rhodopsin (R) to bathorhodopsin phototransformation as an internal standard (phi R----B = 0.67). In contrast to rhodopsin photochemistry, isorhodopsin displays a wavelength dependent quantum yield for photochemical generation of bathorhodopsin at 77 K. Measurements at seven wavelengths yielded values ranging from a low of 0.089 +/- 0.021 at 565 nm to a high of 0.168 +/- 0.012 at 440 nm. An analysis of these data based on a variety of kinetic models suggests that the I----B phototransformation encounters a small activation barrier (approximately 0.2 kcal mol-1) associated with the 9-cis----9-trans excited-state torsional-potential surface. The 9-cis retinal chromophore in solution (EPA, 77 K) has the smallest oscillator strength relative to the other isomers: 1.17 (all-trans), 0.98 (9-cis), 1.04 (11-cis), and 1.06 (13-cis). The effect of conformation is quite different for the opsin-bound chromophores. The oscillator strength of the lambda max absorption band of I is observed to be anomalously large (1.11) relative to the lambda max absorption bands of R (0.98) and B (1.07). The wavelength-dependent photoisomerization quantum yields and the anomalous oscillator strength associated with isorhodopsin provide important information on the nature of the opsin binding site. Various models of the binding site were tested by using INDO-PSDCI molecular orbital theory to predict the oscillator strengths of R, B, and I and to calculate the barriers and energy storage associated with the photochemistry of R and I for each model. Our experimental and theoretical investigation leads to the following conclusions: (a) The counterion (abbreviated as CTN) is not intimately associated with the imine proton in R, B, or I. The counterion lies underneath the plane of the chromophore in R and I, and the primary chromophore-counterion electrostatic interactions involve C15-CTN and C13-CTN. These interactions are responsible for the anomalous oscillator strength of I relative to R and B. (b) The presence of a small activation barrier (~0.2 kcal mol-1) in the 9-cis - 9-trans excited-state surface is associated with the location of the counterion as well as the intrinsic photophysical properties of the 9-cis chromophore. The principal difference between the 1 1-cis -c 1 -transphoto reaction surface and the 9-cis - 9-trans photoreaction surface is the lack of effective electrostatic stabilization of distorted 9 = 10 conformations due to incomplete charge polarization. (c) Hydrogen bonding to the imine proton, ifpresent, does not involve the counterion. We conclude that water in the active site, or secondary interactions with the protein (not involving the CTN), are responsible. (d) All photochemical transformations involve one-bond photoisomerizations.This prediction is based on the observation of a very small excited state barrier for the I -- B photoreaction and a negative barrier for the R - B phototransformation, coupled with the theoretical prediction that all two-bond photoisomerizations have significant S, barriers while one-bond photoisomerizations have small to negative S, barriers.(e) Rhodopsin is energetically stabilized relative to isorhodopsin due to both electrostatic interactions and conformational distortion, both favoring stabilization of R. The INDO-PSDCI calculations suggest that rhodopsin chromophore-CTN electrostatic interactions provide an enhanced stabilization of -2 kcal mol-1 relative to I. Conformational distortion of the 9-cis chromophore-lysine system accounts for -3 kcal mol-1. (f) Energy storage in bathorhodopsin is-60% conformational distortion and 40% charge separation. Our model predicts that the majority of the chromophore protein conformational distortion energy involves interaction of the C,3(-CH3)=CI4--C,5=N-lysine moiety with nearby (unknown) protein residues. (g) Strong interactions between the counterion and the chromophore in R and I will generate weak, but potentially observable charge-transfer bands in the near infrared. The key predictions are the presence of an observable charge-transfer transition at 859 nm (1 1,640 cm- 1) in I and an analogous, but slightly weaker band at 897 nm (11,150 cm-1) in R. Both transitions involve the transfer of an electron from the counterion into low-lying l theta* molecular orbitals.