Functional Study Of The Important Amino Acid Residues D1-N338 And D1-S268 In Photosystem ?

The high-resolution crystal structure of Photosystem II reveals several important hydrogen-bonding networks,which can serve as a pathway for protons to leave or water molecules to enter during the oxidation of photosynthetic water.Among them,D1-N338 and D1-S268 in the D1 protein are important amino acid residues on the hydrogen bond network around the Mn4 Ca O5 clusters in the donor side and the hydrogen bond network around non-heme iron in the acceptor side,respectively.In order to further study their function in the process of water oxidation.Thermophilic cyanobacteria Thermosynechococcus vulcanus is used as the research material,and slected the D1-N338 and D1-S268 sites are selected as the main research objects to construct their mutants by site-directed mutation.The function of the mutants from thermophilic cyanobacteria T.vulcanus characterized by using of spectroscopy,oxygen electrodes,chlorophyll fluorescence decay kinetics et al,revealed that these amino acid residues are important for maintaining normal photosynthetic water oxidation.(The thermophilic cyanobacteria wild type contains three homologous genes that simultaneously encode the D1 protein,namely psb A1?psb A2?psb A3.In the experiment,the psb A1?psb A2 genes are chosed to knock out to obtain WT*(psb A3–Smr),which is referred to as WT* in the following paper.Then,site-directed mutations on the psb A3 gene is performed to obtain various experimental mutants).The main results are as follows:(1)D1-N338 E,D1-N338 F,D1-N338 L,D1-S268 H,D1-S268 N,D1-S268 T mutants from thermophilic cyanobacteria are successfully obtained by site-directed mutation in vivo.(2)The photoautotrophic growth status of the mutants is determined and showed that all D1-N338 F,D1-N338 E,D1-N338 L,D1-S268 H,and D1-S268 N mutants exhibits almost the same growth rate as that of the wild type control WT* under the white light with an inteisity of 40 ?mol m-2 s-1,while the photoautotrophic growth rate of the mutant D1-S268 H and D1-S268 T cells are slightly lower than that of the WT*.(3)The oxygen evolving activities of D1-N338 and D1-S268 mutant cells are measured.At p H = 6.5,the oxygen-evolving activities of N338 E,N338F,and N338 L are about 98%,91%,and 88% of that measured from WT*,respectively.The oxygenevolving activities of S268 H,S268N,and S268 T are about 92%,73%,and 76% of that of WT*,respectively,indicate that both the D1-N338 site and D1-S268 sites are important to maintain normal oxygen evolving activities.(4)Absorption spectra measured at room temperature and 77 K fluorescence spectra excited at 436 nm and 570 nm are performed,respectively.The results showed that mutant N338 F exhibits a significantly different behavior in the energy transfer from phycobilisome to photosystem core compared to that of with other mutants and the WT* cell.(5)QA-reoxidized chlorophyll fluorescence decay kinetics of the D1-N338 and D1-S268 mutants are measured in the absence of DCMU and presence of DCMU,and the results showed that mutantations of D1-N338 F and D1-N338 L altered the electron transfer in the donor side,while the elctron transfer in the acceptor side is the same as that of the WT* in all mutants.In summary,the mutations at the D1-N338 and D1-S268 sites affected the oxygen evolving activity indicate that these sites are very important for maintaining normal photosynthetic function.The functional changes caused by site mutations are likely to be due to changes in the hydrogen bond network structure around the PSII donor and acceptor sides,respectively.