Resonance raman analysis of chromophore structure in the lumi-R photoproduct of phytochrome.
Resonance Raman vibrational spectra of the Pr, lumi-R, and Pfr 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 Pr 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 Pr is converted to lumi-R, new bands appear in the C = C and C = N 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 C = N stretching mode marker band shifts from 1576 cm-1 in Pr to 1569 cm-1 in lumi-R and to 1552 cm-1 in Pfr. Normal mode calculations show that the frequency drop of this band in the lumi-R-->Pfr interconversion is an indication of a C14-C15 syn-->anti conformational change. Moderately intense hydrogen out-of-plane modes that occur at 805 cm-1 in Pr 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 Pfr. These observations indicate that the C and D rings of the chromophore in Pr and lumi-R are moderately planar but that they become highly distorted in Pfr. This information suggests that the primary photochemistry in phytochrome is a Z-->E isomerization of the C15 = C16 bond of Pr giving lumi-R. This is followed by a thermal syn-->anti C14-C15 conformational relaxation to form Pfr. A four-state model is presented to explain the chromophore structural changes in Pr, lumi-R, and Pfr that uses hydrogen bonding to the surrounding protein to stabilize the high-energy Pfr C15 = C16, C14-C15, E,anti chromophore structure. This implicates an anchor and release mechanism between the chromophore and protein that might lead to altered biological signaling in the plant.
Andel
F
F
Department of Chemistry, University of California, Berkeley 94720, USA.
Lagarias
J C
JC
Mathies
R A
RA
D006860:Hydrogen Bonding; D007536:Isomerism; D008027:Light; D008958:Models, Molecular; D010777:Photochemistry; D010834:Phytochrome / Q000737:chemistry* / Q000302:isolation & purification / Q000528:radiation effects; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman / Q000379:methods
D010834:Phytochrome