Resonance Raman Analysis of Chromophore Structure in the Lumi-R Photoproduct of Phytochrome

Abstract

Resonance Raman vibrational spectra of the P_r, lumi-R, and P_fr forms of phytochrome have been obtained using low-temperature trapping and room temperature flow techniques in conjunction with shifted-excitation Raman difference spectroscopy (SERDS). The P_r to lumi-R photoconversion exhibits a thermal barrier and is completely blocked at 30 K, indicating that thermally assisted protein relaxation is necessary for the primary photochemistry. When P_r is converted to lumi-R, new bands appear in the CC and CN stretching regions at 1651, 1636, 1590, and 1569 cm^-1, indicating that a significant structural change of the chromophore has occurred. The photoconversion also results in an 18 cm^-1 decrease in the N−H rocking band in lumi-R. Normal mode calculations correlate this frequency drop with a change in the geometry of the C₁₅ methine bridge of the phytochromobilin chromophore. Additionally, a CN stretching mode marker band shifts from 1576 cm^-1 in P_r to 1569 cm^-1 in lumi-R and to 1552 cm^-1 in P_fr. Normal mode calculations show that the frequency drop of this band in the lumi-R → P_fr interconversion is an indication of a C₁₄−C₁₅syn → anti conformational change. Moderately intense hydrogen out-of-plane modes that occur at 805 cm^-1 in P_r shift to 829 and 847 cm^-1 upon photoconversion to lumi-R and are replaced by a very intense mode at 814 cm^-1 in P_fr. These observations indicate that the C and D rings of the chromophore in P_r and lumi-R are moderately planar but that they become highly distorted in P_fr. This information suggests that the primary photochemistry in phytochrome is a Z → E isomerization of the C₁₅C₁₆ bond of P_r giving lumi-R. This is followed by a thermal syn → anti C₁₄−C₁₅ conformational relaxation to form P_fr. A four-state model is presented to explain the chromophore structural changes in P_r, lumi-R, and P_fr that uses hydrogen bonding to the surrounding protein to stabilize the high-energy P_fr C₁₅C₁₆, C₁₄−C₁₅, E,anti chromphore structure. This implicates an anchor and release mechanism between the chromophore and protein that might lead to altered biological signaling in the plant.