Time-resolved resonance Raman and quantum yield studies of rhodopsin photochemistry

The primary event in vision is the ultrafast, light-driven cis -to-trans isomerization of the 11-cis retinal chromophore in the visual pigment rhodopsin. How can we understand the mechanism and structural dynamics of this unique photoisomerization that initiates all vertebrate and invertebrate vision? This dissertation describes how I have used resonance Raman spectroscopy and studies of the reaction quantum yield to unravel the dynamics of the light-induced isomerization and in particular, the structural evolution of the chromophore on the picosecond time scale. Raman spectra of the initial photoproduct, photorhodopsin, provide the first evidence that the chromophore structure formed in <1 ps has a distorted, all-trans configuration. The presence of intense anti-Stokes peaks reveals that photorhodopsin is initially very hot, with low-frequency vibrational temperatures of >2000 K. The photoproduct cools by intramolecular vibrational energy redistribution (IVR) in ∼3 ps to form the bathorhodopsin intermediate. The similar frequencies of the ethylenic, hydrogen out-of-plane, and fingerprint modes in bathorhodopsin and photorhodopsin show that the all-trans chromophore is common to both species. Energy randomization by IVR on the picosecond time scale indicates that energy storage in the primary visual photoproduct is complete in less than 3 ps.; A new transient intermediate is identified in Stokes Raman experiments which sheds light on the structure and dynamics of the photoexcited rhodopsin molecules that fail to isomerize. Unique Raman peaks at 290, 992, 1254, 1290 and 1569 cm-1 are observed only in the 0 ps delay spectrum and are attributed to an unreactive excited-state species based on the observed lifetime of <700 fs. The fingerprint and ethylenic regions are significantly altered relative to those of ground-state rhodopsin; in particular the 1569 cm-1 ethylenic mode is unusually high in frequency, implying that the retinal chromophore in this transient may be conformationally distorted and have a more localized electronic structure.; The excited-state dynamics of rhodopsin have also been probed through accurate measurements of the photoreaction quantum yield. A 5% decrease in quantum yield over the incident wavelength range of 500 to 570 nm disproves the ∼60 year old belief that the quantum yield for visual photochemistry is wavelength independent. A vibronic analysis of the absorption spectrum coupled with a Landau-Zener model for the reactive surface-crossing process successfully explains the experimentally observed trend. This calculation shows that the relative partitioning of energy into reactive and unreactive vibrations depends on incident wavelength, and that minimally nine delocalized torsional modes must be included in the reaction coordinate. My analysis of the quantum yield in terms of reactive and unreactive vibrational modes, together with their direct observation in the picosecond Raman studies, significantly enhances our mechanistic understanding of the primary event in vision.