MUTATIONAL ANALYSIS OF THE PEA PHYTOCHROME-A CHROMOPHORE POCKET - CHROMOPHORE ASSEMBLY WITH APOPHYTOCHROME-A AND PHOTOREVERSIBILITY

Ten site-specific mutants of pea apophytochrome A were expressed in Sa ccharomyces cerevisiae and analyzed for chromophore assembly with apop rotein and photoreversible absorbance changes. The mutants constitute two specific changes for each of five conserved amino acid residues lo cated in the microenvironment of the chromophore attachment residue, w hich is Cys-323 in pea phytochrome A. All mutant apophytochromes were autocatalytically able to covalently attach phycocyanobilin, indicatin g that there were no major structural perturbations in the apoproteins . However, the rate of chromophore ligation varied significantly among the mutants. Spectrally, the mutant holophytochromes are of three typ es: mutant phytochromes that are indistinguishable from the wild-type adduct, mutants with blue-shifted P(r) and P(fr) absorption maxima com pared to the wild-type adduct, and mutants that are not photoreversibl e. From an analysis of the results, we concluded that the residues Asp -309, Arg-318, His-321, and Gln-326 are probably not catalytically inv olved in the chromophore ligation reaction, but some residues may play significant structural and stereochemical roles. Arg-318 might anchor the chromophore, as has been suggested [Partis, M.D., & Grimm, R. (19 90) Z. Naturforsch. 45c, 987-998, Parker, W., et al. (1993) Bioconjuga te Chem. (in press)]. The conserved Gln-326, three residues downstream from the chromophore attachment site, is not electrostatically critic al for the spectral integrity and photoreversibility of phytochrome, b ut this residue is sterically important to the lyase activity. It appe ars that the role of the five amino acid residues in the N- and C-term inal vicinities of the chromophore binding Cys-323 is structural rathe r than catalytic for the ligation reaction.