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-tempera ture bacteriorhodopsin (BR) photocycle, is measured via picosecond time-res olved coherent anti-Stokes Raman spectroscopy (PTR/CARS). An examination of the excitation conditions and BR photocycle kinetics, as well as the vibra tional 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 resulta nt background-free (Lorentzian line shapes) CARS spectrum contains 24 disti nct vibrational features which provide the most complete structural charact erization of J-625 yet reported. Comparisons of the 5-625 vibrational spect rum 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 sign ificantly different structure than that of K-590. Specifically, 5-625 has ( i) an all-trans retinal configuration, (ii) increased electron density in t he C=C stretching modes as manifested by increased C=C stretching frequenci es relative to those in both BR-570 and K-590, (iii) significant delocalize d hydrogen out-of-plane motion not observed in any other BR species, (iv) d ecreased C-CH3 in-plane wagging motion, and (v) a Schiff-base bonding envir onment 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 inter mediate found in the photoreaction of the artificial BR pigment, BR5.12, co ntaining a five-membered ring spanning the C12C13=C-14 bonds (thereby block ing C-13=C-14 isomerization), support the conclusion that the J-625 structu re 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-tra ns retinal, C-13=C-14 isomerization cannot be the primary reaction coordina te 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 chan ges in the retinal structural and/or retinal-protein interactions must comp rise the primary reaction coordinates that precede C-13=C-14 isomerization. These results require that significant changes in the mechanistic model de scribing the room-temperature BR photocycle be considered.