Vibrational Spectrum of the J-625 Intermediate in the Room Temperature Bacteriorhodopsin Photocycle

The vibrational spectrum (800-1700 cm(-1) region) of the 5-625 intermediate, formed within 200-500 fs (3.5 ps decay time to K-590) in the room-temperature bacteriorhodopsin (BR) photocycle, is measured via picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). An examination of the excitation conditions and BR photocycle kinetics, as well as the vibrational CARS data, convincingly demonstrates that these PTR/CARS spectra can be quantitatively analyzed in terms of primarily BR-570 and J-625 by using third-order nonlinear susceptibility (chi((3))) relationships. The resultant background-free (Lorentzian line shapes) CARS spectrum contains 24 distinct vibrational features which provide the most complete structural characterization of J-625 yet reported. Comparisons of the 5-625 vibrational spectrum with those of groundstate BR-570 and the K-590 intermediate show that J-625 maintains some structural similarities with BR-570 while it has a significantly different structure than that of K-590. Specifically, 5-625 has (i) an all-trans retinal configuration, (ii) increased electron density in the C=C stretching modes as manifested by increased C=C stretching frequencies relative to those in both BR-570 and K-590, (iii) significant delocalized hydrogen out-of-plane motion not observed in any other BR species, (iv) decreased C-CH3 in-plane wagging motion, and (v) a Schiff-base bonding environment similar to that of BR-570 and distinctively different from that in K-590. Comparisons between the PTR/CARS spectra of J-625 and T5.12, an intermediate found in the photoreaction of the artificial BR pigment, BR5.12, containing a five-membered ring spanning the C12C13=C-14 bonds (thereby blocking C-13=C-14 isomerization), support the conclusion that the J-625 structure reflects the reaction coordinates in the BR photocycle that precede C13=C14 isomerization. Since these PTR/ CARS data show J-625 to have an all-trans retinal, C-13=C-14 isomerization cannot be the primary reaction coordinate described in numerous models for the BR photocycle. The all-trans to 13-cis isomerization must occur as 5-625 transforms into K-590, and other changes in the retinal structural and/or retinal-protein interactions must comprise the primary reaction coordinates that precede C-13=C-14 isomerization. These results require that significant changes in the mechanistic model describing the room-temperature BR photocycle be considered.