Fourier-Transform Infrared Spectroscopy of Phytochrome:  Difference Spectra of the Intermediates of the Photoreactions

The photocycle of 124 kDa phytochrome A from Avena sativa was studied by Fourier-transform infrared spectroscopy at low temperatures. Difference spectra between the parent state Pr and the intermediates of the Pr-->Pfr pathway, i.e. lumi-R, meta-Ra, and meta-Rc, and between Pfr and the intermediates of the Pfr-->Pr pathway, lumi-F and meta-F, were obtained in 1H2O and 2H2O for the first time. Each spectrum shows characteristic spectral features which allow a clear distinction between the different intermediates. A general feature is that greater changes occur with increasing temperature, i.e. at the later steps of the photoreactions. Nevertheless, the changes in the spectral regions of the protein (amide I and amide II) were found to be surprisingly small, excluding larger conformational changes of the protein. All spectra of the intermediates are characterized by a strong negative band around 1700 cm-1. This band is tentatively assigned to the C = O stretch of ring D of the chromophore. Since it is not observed in the difference spectra between the parent states, it is concluded that ring D is located in a similar molecular environment in Pr and Pfr. In the photoproducts lumi-R and lumi-F, this band undergoes an upshift to 1720 cm-1. The high frequencies suggest that the chromophore is protonated in these intermediates as well as in Pr and Pfr.