Vibrational spectrum of a picosecond intermediate in the artificial BR5.12photoreaction: Picosecond time-resolved CARS of T5.12

The vibrational spectrum of an intermediate, T5.12, in the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a five-membered ca rbon ring spanning the C-12-C-13=C-14 bonds (BR5.12) is measured by picosec ond time-resolved coherent anti-Stakes Raman spectroscopy (PTR/CARS). Obser ved initially by picosecond transient absorption (PTA) measurements, T5.12 is the only intermediate in the BR5.12 photoreaction (i.e., T5.12 decays on ly to BR5.12). BR5.12 does not have a photocycle analogous to that in nativ e BR, presumably because the five-membered ring blocks the reaction coordin ate leading to C-13=C-14 bond isomerization. Since T5.12 may therefore repr esent the molecular events (reaction coordinates) that precede C-13=C-14 bo nd isomerization, its vibrational spectrum may aid in elucidating the prima ry reaction coordinate(s) in the BR photocycle. Although T5.12 is identifie d via a red-shifted absorption (660 nm maximum. <3 ps formation with 3 ps B R5.12 excitation and decay in 17 +/- 1 ps), no spectroscopic data which dir ectly characterize the retinal structure in T5.12. and thereby the role of bonding changes, have been available. The PTR/CARS vibrational data present ed here show that T5.12 contains (i) an all-trans retinal configuration, (i i) significant hydrogen out-of-plane motion localized in specific normal mo des, (iii) increased pi-electron density in the C=C stretching modes manife sted by frequency increases, (iv) restricted in-plane C-CH3 rocking motion, and (v) a Schiff-base environment similar to that in BR5.12. These PTR/CAR S data also confirm that T5.12 decays exclusively to BR5.12. The vibrationa l spectrum of T5.12 makes it evident that no complete C=C isomerization nor C-C rotation in any retinal bond occurs upon excitation of BR5.12. The exc ellent agreement between the kinetic lifetime of T5.12 (from PTA and PTR/CA RS data) and its stimulated emission Lifetime suggests that T5.12 may be an excited electronic state. In such a case, the PTR/CARS data presented here are the first to be reported from an excited electronic state of a protein . Regardless of whether T5.12 is an excited or ground electronic state, the vibrational spectra of T5.12 reflect the retinal structure(s) that precede s C-13=C-14 bond isomerization in the BR photocycle. The relevance of T5.12 PTR/CARS data to the native BR photocycle is discussed in terms of the int ermediates K-590, J-625, and I-460. Direct analyses of the respective vibra tional spectra of T5.12 and K-590 demonstrate that they contain distinctly different retinal structures, but since no vibrational data assignable dire ctly to either I-460 or J-625 have been reported, comparisons of T5.12 with these intermediates are based only on analogy. Comparisons of the vibratio nal spectra of T5.12 and native BR intermediates independently provide insi ght into the structural changes in retinal that could occur prior to C-13=C -14 bond isomerization in native BR.