FTIR Studies of Phytochrome Photoreactions Reveal the CO Bands of the Chromophore: Consequences for Its Protonation States, Conformation, and Protein Interaction

Abstract

The molecular changes of phytochrome during red → far-red and reverse photoreactions have been monitored by static infrared difference spectroscopy using the recombinant 65 kDa N-terminal fragment assembled with a chromophore chemically modified at ring D or with a chromophore isotopically labeled with ¹⁸O at the carbonyl group of ring A. This allows the identification of the CO stretching vibrations of rings D and A. We exclude the formation of an iminoether in Pfr. The positions of both these modes show that the chromophore always remains protonated. The upshift of the CO stretch of ring D in the first photoproducts is explained by a twisted methine bridge connecting rings C and D. The changes in the vibrational pattern during the red → far-red conversion show that the backreaction is not just the reversal of the forward reaction. The infrared difference spectra of the fragment deviate very little from those of the full-length protein. The differences which are related to the lack of the C-terminal half of the protein constituting the signaling domain are possibly important for the understanding of the signaling mechanism.