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 pastor is and assembled into two chromopeptides with the chromophores phytochromobilin (P Phi B) and phycocyanobilin (PCB), respectively, The P-r and P-fr 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 P Phi B 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 P Phi B- and PCB-assembled phytochromes. Only in the P-r state may the RR spectral changes also reflect subtle differences of the P Phi B and PCB conformations in the 65-kDa phyA, presumably brought about by the specific steric requirements of the vinyl and ethyl groups. (C) 1997 Federation of European Biochemical Societies.