Effect of chromophore exchange on the resonance Raman spectra of recombinant phytochromes.
The recombinant 65-kDa polypeptide of phyA oat phytochrome was expressed by yeast Pichia pastoris and assembled into two chromopeptides with the chromophores phytochromobilin (PphiB) and phycocyanobilin (PCB), respectively. The Pr and Pfr states of the two protein variants were characterized by resonance Raman (RR) spectroscopy and compared with native phyA oat phytochrome demonstrating that the deletion of the C-terminal half of phyA does not alter the structure of the chromophore site within the N-terminal half. Most of the RR spectral changes observed upon replacing PphiB by PCB can be attributed exclusively to altered vibrational mode compositions due to the different ring D substitutions (vinyl vs. ethyl), implying that the chromophore structures are largely the same for PphiB- and PCB-assembled phytochromes. Only in the Pr state may the RR spectral changes also reflect subtle differences of the PphiB and PCB conformations in the 65-kDa phyA, presumably brought about by the specific steric requirements of the vinyl and ethyl groups.