Differential effects of mutations in the chromophore pocket of recombinantphytochrome on chromoprotein assembly and P-r-to-P-fr photoconversion

Site-directed mutagenesis was performed with the chromophore-bearing N-term inal domain of oat phytochrome A apoprotein (amino acid residues 1-595). Ex cept for Trp366, which was replaced by Phe (W366F), all the residues exchan ged an in close proximity to the chromophore-binding Cys321 (i.e. P318A, P3 18K, H319L, S320K, H322L and the double mutant L323R/Q324D). The mutants we re characterized by their absorption maxima, and the kinetics of chromophor e-binding and the P-r-->P-fr conversion. The strongest effect of mutation o n the chromoprotein assembly, leading to an almost complete loss of the chr omophore binding capability, was found for the exchanges of His322 by Leu ( H322L) and Pro318 by Lys (P318K), whereas a corresponding alanine mutant (P 318A) showed wild-type behavior. The second histidine (H319) is also involv ed in chromophore fixation, as indicated by a slower assembly rate upon mut ation (H319L). For the other mutants, an assembly process very similar to t hat of the wild-type protein was found. The light-induced P-r-->P-fr conver sion kinetics is altered in the mutations H319L and S320K and in the double mutant L323R/Q324D, all of which exhibited a significantly faster I-700 de cay and accelerated P-fr formation. P318 is also involved in the P-r-->P-fr conversion, the millisecond steps (formation of P-fr) being significantly slower for P318A. Lacking sufficient amounts of W366F, assembly kinetics co uld not be determined in this case, while the fully assembled mutant underw ent the P-r-->P-fr conversion with kinetics similar to wild-type protein.