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Time-Resolved Resonance Raman Structural Studies of the pB' Intermediate in the Photocycle of Photoactive Yellow Protein

Duohai Pan ^*, Andrew Philip , Wouter D. Hoff  and Richard A. Mathies ^*

^* Department of Chemistry, University of California at Berkeley, Berkeley, California 94720; and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637

Correspondence: Address reprint requests to Richard A. Mathies, Tel.: 510-642-4192; Fax: 510-642-3599; E-mail: rich{at}zinc.cchem.berkeley.edu.

Time-resolved resonance Raman spectroscopy is used to obtain chromophore vibrational spectra of the pR, pB', and pB intermediates during the photocycle of photoactive yellow protein. In the pR spectrum, the C₈–C₉ stretching mode at 998 cm^-1 is 60 cm^-1 lower than in the dark state, and the combination of C–O stretching and C₇H=C₈H bending at 1283 cm^-1 is insensitive to D₂O substitution. These results indicate that pR has a deprotonated, cis chromophore structure and that the hydrogen bonding to the chromophore phenolate oxygen is preserved and strengthened in the early photoproduct. However, the intense C₇H=C₈H hydrogen out-of-plane (HOOP) mode at 979 cm^-1 suggests that the chromophore in pR is distorted at the vinyl and adjacent C₈–C₉ bonds. The formation of pB' involves chromophore protonation based on the protonation state marker at 1174 cm^-1 and on the sensitivity of the COH bending at 1148 cm^-1 as well as the combined C–OH stretching and C₇H=C₈H bending mode at 1252 cm^-1 to D₂O substitution. The hydrogen out-of-plane Raman intensity at 985 cm^-1 significantly decreases in pB', suggesting that the pR-to-pB' transition is the stage where the stored photon energy is transferred from the distorted chromophore to the protein, producing a more relaxed pB' chromophore structure. The C=O stretching mode downshifts from 1660 to 1651 cm^-1 in the pB'-to-pB transition, indicating the reformation of a hydrogen bond to the carbonyl oxygen. Based on reported x-ray data, this suggests that the chromophore ring flips during the transition from pB' to pB. These results confirm the existence and importance of the pB' intermediate in photoactive yellow protein receptor activation.

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