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Fourier-transform resonance Raman-spectroscopy of intermediates of the phytochrome photocycle
Citation key ISI:A1995RR46700023
Author Matysik, J and Hildebrandt, P and Schlamann, W and Braslavsky, S and Schaffner, K
Pages 10497-10507
Year 1995
ISSN 0006-2960
Address PO BOX 57136, WASHINGTON, DC 20037-0136
Journal Biochemistry
Volume 34
Number 33
Month AUG 22
Abstract The parent states of the 124-kDa phytochrome (phy A from Avena sativa) and intermediates of its photocycle were studied by low-temperature Fourier-transform resonance Raman spectroscopy: Spectra of the primary photoproducts I-700 and lumi-F and of the thermal intermediate meta-F have been obtained for the first time. The spectra of the stable photochromic forms of photochrome, P-r and P-fr, presented in this work are significantly better in signal-to-noise ratio and resolution than previously published spectra, demonstrating the distinct advantages of our experimental approach. The high spectral quality allows for the identification of subtle details of the vibrational band pattern so that the resonance Raman spectra, which have been measured from samples in H2O and D2O, constitute a solid basis for the structural analysis of the various forms of phytochrome. Notwithstanding the current uncertainty in the vibrational assignment of many resonance Raman bands, the spectral changes of the tetrapyrrole chromophore can plausibly be interpreted in terms of conformational changes at two different methine bridges, i.e., torsions around two single bonds and the E/Z isomerization of a double bond. Within the framework of this interpretation, which is based on a vibrational analysis of biliverdin dimethyl ester (Smit, K., Matysik, J., Hildebrandt, P., and Mark, F. (1993) J. Phys. Chem. 97, 11887-11900), a consistent model is proposed to describe the molecular events in the chromophore during the photocycle. The involvement of a proton transfer in the primary photoprocess of P, can safely be ruled out. However, previous conclusions concerning the chromophore protonation in the individual states appear premature at the present state of the vibrational assignment. In particular, the attribution of a broad band at 1100 (-1) cm to the N-H out-of-plane bending of the protonated pyrrolenin nitrogen (Hildebrandt, P., Hoffmann, A., Lindemann, P., Heibel, G., Braslavsky, S. E., Schaffner, K., & Schrader, B. (1992) Biochemistry 32, 7957-7962) has now been found to be erroneous.
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