Probing the photoreaction mechanism of phytochrome through analysis of resonance Raman vibrational spectra of recombinant analogues

Resonance Raman spectra of native and recombinant analogues of oat phytochrome have been obtained and analyzed in conjunction with normal mode calculations. On the basis of frequency shifts observed upon methine bridge deuteration and vinyl and C-15-methine bridge saturation of the chromophore, intense Raman lines at 805 and 814 cm(-1) in P-r and P-fr, respectively, are assigned as C-15-hydrogen out-of-plane (HOOP) wags, lines at 665 cm(-1) in P-r and at 672 and 654 cm(-1) in P-fr are assigned as coupled C=C and C-C torsions and in-plane ring twisting modes, and modes at approximate to 1300 cm(-1) in P-r are coupled N-H and C-H rocking modes. The empirical assignments and normal mode calculations support proposals that the chromophore structures in P-r and P-fr are C-15-Z,syn and C-15-E,anti, respectively. The intensities of the C-15-hydrogen out-of-plane, C=C and C-C torsional, and in-plane ring modes in both P-r and P-fr suggest that the initial photochemistry involves simultaneous bond rotations at the C-15-methine bridge coupled to C-15-H wagging and D-ring rotation. The strong nonbonded interactions of the C- and D-ring methyl groups in the C-15-E,anti P-fr chromophore structure indicated by the intense 814 cm(-1) C-15 HOOP mode suggest that the excited state of P-fr and its photoproduct states are strongly coupled.