The analysis of site-directed mutants in the glutamate 189 and histidine 190 in the D1 protein of photosystem II

Abstract

The primary charge separation in photosystem (PS) II takes place within 3 ps, generating highly oxidative P680+. Site-directed mutagenesis has been proven to be a valuable tool for elucidating the function of each amino acid residue in PSII. Several mutants at glutamate-189 and histidine-190 in the putative cd-helix of the D1 protein were constructed in Chlamydomonas reinhardtii, and the effects of mutation on PS II functions were studied. In E189Q and E189L mutants, they were able to grow photoautotrophycally, but the oxygen evolution activity decreased to a half of the wild type. On the other hand, E189D and H190R mutants lost the oxygen evolution activity, which led to the non-photoautotrophical phenotype. In order to obtain further insights on the effects of the mutations, two-dimensional fluorescence photon counting experiments were carried out. The results showed that the fluorescence lifetime at 77K was markedly prolonged in all mutants, suggesting that the yield of the charge separation becomes to be lower due to the decrease in redox potential of P680 and/or Yz. In PS II reaction center, isolated from His-tagged versions of these mutants, the activity of pheophytin photoacummulation was reduced to 60-70 % of the control. Therefore, it is indicated that these two amino acids exist in the close vicinity of P680 and play important role(s) in the charge separation, in addition to their involvement to the donor side function as revealed E189D and H190R mutants.