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

Abstract

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 C 8–C 9 stretching mode at 998 cm −1 is ∼60 cm −1 lower than in the dark state, and the combination of C–O stretching and C 7H = C 8H bending at 1283 cm −1 is insensitive to D 2O 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 C 7H = C 8H hydrogen out-of-plane (HOOP) mode at 979 cm −1 suggests that the chromophore in pR is distorted at the vinyl and adjacent C 8–C 9 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 C 7H = C 8H bending mode at 1252 cm −1 to D 2O 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.