Gh. Atkinson et al., Vibrational spectrum of the J-625 intermediate in the room temperature bacteriorhodopsin photocycle, J PHYS CH A, 104(18), 2000, pp. 4130-4139
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.