The nature of the primary photochemical events in rhodopsin and isorhodopsin

• Published in: Biophysical journal Vol. 53; no. 3; pp. 367 - 385
• Main Authors: Birge, R.R., Einterz, C.M., Knapp, H.M., Murray, L.P.
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 01.03.1988; Biophysical Society; The Biophysical Society
• Subjects: Binding Sites; Biological and medical sciences; Calorimetry; Eye Proteins; Fundamental and applied biological sciences. Psychology; Hydrogen Bonding; Ions; Molecular biophysics; Photochemistry; Photochemistry. Photosynthesis. Bioluminescence; Radiation-biomolecule interaction; Retinal Pigments; Retinaldehyde; Rhodopsin; Rod Opsins; MO method; Absorption spectrometry; Rhodopsin; Photoisomerization; Primary reaction; Vision; Low temperature; Photochemistry
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(88)83114-X

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.