LUMENAL SIDE HISTIDINE MUTATIONS IN THE D1-PROTEIN OF PHOTOSYSTEM-II AFFECT DONOR SIDE ELECTRON-TRANSFER IN CHLAMYDOMONAS-REINHARDTII

Site-directed mutants of the D1 protein generated in Chlamydomonas rei nhardtii have been characterized to determine whether specific lumenal side histidine residues participate in or directly influence electron transfer. Histidine 195 (H195), a conserved residue located near the amino-terminal end of the D1 transmembrane alpha-helix containing the putative P680 chlorophyll ligand H198, was changed to asparagine (H195 N), aspartic acid (H195D), and tyrosine(H195Y). These H195 mutants dis played essentially wild-type rates of electron transfer from the water -oxidizing complex to 2,6-dichlorophenolindophenol. Flash-induced chlo rophyll a (Chl a) fluorescence yield rise and decay measurements for M n-depleted membranes of the H195Y and H195D mutants, however, revealed modified Y-Z to P680(+) electron transfer kinetics. The rate of the v ariable Chl a fluorescence rise was reduced approximately 10-fold in H 195Y and H195D relative to the wild type. In addition, the rate of Chl a fluorescence decay in the presence of 3-(3,4-dichlorophenyl)-1,1-di methylurea was approximately 50-fold more rapid in H195D than in the w ild type. These results can be accommodated by a change in the midpoin t potential of Y-Z(+)/Y-Z which is apparent only upon the removal of t he Mn cluster. In addition, we have generated a histidine to phenylala nine substitution at histidine 190 (H190), a conserved residue located near the lumenal thylakoid surface of D1 in close proximity to the se condary donor Y-Z. The H190F mutant is characterized by an inability t o oxidize water associated with the loss of the Mn cluster and severel y altered donor side kinetics. These and other results suggest that H1 90 may participate in redox reactions leading to the assembly of the M n cluster.