Ultraviolet resonance Raman spectra of pea intact, large, and small phytochromes: Differences in molecular topography of the red- and far-red-absorbing forms
Ultraviolet resonance Raman (UV RR) spectra excited at 244 nm were observed for pea intact, large, and small phytochromes at pH 7.8. Raman bands assignable to Trp residues dominated the UV RR spectra. The intensity ratios of Trp W7 doublet bands, I(1358)/I(1342), of all three phytochromes in the red light-absorbing form (Pr) were almost the same as that of an aqueous Trp solution, indicating that most of the six and four Trp residues in the 59-kDa chromophoric and the C-terminal 59-kDa nonchromophoric domains, respectively, reside in hydrophilic microenvironments in Pr. This ratio increased under red light illumination, where photoequilibria are attained between Pr and the far-red-absorbing form (Pfr) for intact and small phytochromes and among Pr, a bleached intermediate (Ibl), and Pfr for large phytochromes. The increase of the intensity ratio was most prominent for small phytochromes. These observations suggest that the microenvironments around some Trp residues become more hydrophobic due to conformational changes induced by phototransformation from Pr to Ibl and that the hydrophobicity increase occurs mainly in the chromophoric domain. Among the six Trp residues in the chromophoric domain, Trp365 and Trp567 are likely candidates for those involved in this hydrophobicity increase. The intensity distribution of the amide I band shows little beta-sheet in both Pr and Pfr of the intact, large, and small phytochromes and indicates that alpha-helices and nonregular structure are less populated in the chromophoric domain than in the N-terminal 6-kDa segment and the C-terminal nonchromophoric domain.