| Photosystem II, a pigment-protein complex located in the thylakoids of oxygen-evolving plants and algae, is the site of photosynthetic water oxidation. The primary reactions of light-induced electron transfer in photosystem II, initiated by excitation of the primary donor, P680, occur within the reaction center core formed by the D1 and D2 protein subunits. In this work, site-directed mutants of the D1 protein generated in the green alga Chlamydomonas reinhardtii have been characterized to determine whether specific lumenal side histidine residues mediate donor side electron transfer. Utilizing a chloroplast DNA cotransformation system, histidine 195 (H195), a conserved residue located near the amino terminal end of the D1 transmembrane {dollar}alpha{dollar}-helix IV, was changed to asparagine (H195N), aspartic acid (H195D), and tyrosine(H195Y). Despite essentially wild type rates of electron transfer in steady state assays, flash-induced chlorophyll a fluorescence rise and decay measurements for Mn-depleted, non-oxygen-evolving membranes of the H195Y and H195D mutants in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea revealed modified Y{dollar}sb{lcub}rm Z{rcub}{dollar} to P{dollar}sb{lcub}680{rcub}sp+{dollar} electron transfer kinetics. A histidine to phenylalanine substitution was generated at histidine 190 (H190), a conserved residue located near the lumenal thylakoid surface of D1 nearby the secondary donor Y{dollar}sb{lcub}rm Z{rcub}{dollar}. The H190F mutant was characterized by an inability to oxidize water associated with the loss of the Mn cluster and severely altered donor side kinetics. A kinetic spectrum of a flash-induced transient EPR signal from the H190F mutant was identical in linewidth (18-20G) and structure to the wild type spectrum from oxidized Y{dollar}sb{lcub}rm Z{rcub}{dollar}, indicating that unlike the situation on the D2 side, a hydrogen bond between Y{dollar}sb{lcub}rm Z{rcub}{dollar} and H190 on the D1 protein is unlikely. The amplitudes of the A{dollar}sb{lcub}rm T{rcub}{dollar} thermoluminescence bands measured for the H195N, H195Y, H195D mutants, were lower in anplitude than wild type, but the peak temperatures of the bands were not altered. From these data it was concluded that neither Y{dollar}sb{lcub}rm Z{rcub}{dollar} nor H195 was likely to be the A{dollar}sb{lcub}rm T{rcub}{dollar} positive charge component. The A{dollar}sb{lcub}rm T{rcub}{dollar} band was completely abolished in the H190F mutant, either due to the removal of a redox-active histidine or to the modified donor side kinetics. |
