PubMed1997
Bicarbonate anions have a strong positive influence on the electron and proton transfers in photosystem II (PS II). It has been suggested that bicarbonate binds to the non-heme iron and the QB binding niche of the PS II reaction center. To investigate the potential amino acid binding environment of bicarbonate, an arginine residue (R269) of the D1 protein of PS II of Chlamydomonas reinhardtii was mutated into a glycine; our characterization of the resultant mutant (D1-R269G) shows that both the TyrD+ and QA- Fe2+ EPR signals are substantially reduced and assembly of the tetranuclear Mn is lost (R.S. Hutchison, J. Xiong, R.T. Sayre, Govindjee, Biochim. Biophys. Acta 1277 (1996) 83-92). In order to understand the molecular implications of this mutation on the electron acceptor side of PS II, we used chlorophyll (Chl) a fluorescence as a probe of PS II structure and function, and herbicide binding as a probe for changes in the QB binding niche of PS II. Chl fluorescence measurements with the heterotrophically grown D1-R269G mutant cells (or thylakoids), as compared to that of the wild type, show that: rate of electron transfer from QA to the plastoquinone pool, measured by flash-induced Chl a fluorescence decay kinetics, is reduced by - 17 fold; the minimum Chl a fluorescence yield when all QA- is oxidized, is elevated by 2 fold; the level of stable charge separation as inferred from variable Chl fluorescence is reduced by 44%; binary oscillation pattern of variable Chl a fluorescence obtained after a series of light flashes is absent, indicative of the loss of functioning of the two-electron gate on the PS II acceptor side; 77 K PS II Chl a fluorescence emission bands (F685 and F695) are reduced by 20-30% (assuming no change in the PS I emission band). Thermoluminescence data with thylakoids show the absence of the S2QA- and S2QB- bands in the mutant. Herbicide 14C-terbutryn binding measurements, also with thylakoids, show that the QB niche of the mutant is significantly modified, at least 7-8 fold increased terbutryn dissociation constant is shown (220 nM in the mutant versus 29 nM in the wild type); the PS II sensitivity to bicarbonate-reversible formate inhibition is reduced by 5 fold in the mutant, although the formate/bicarbonate binding site still exists in the mutant. This suggests that D1-R269 must play some role in the binding niche of bicarbonate. On the basis of the above observations, we conclude that the D1-R269G mutation has not only altered the structure and function of PS II (QB niche being abnormal), but may also have a decreased net excitation energy transfer from the PS II core to the reaction center and/or an increased number of inactivated reaction center II. We also discuss a possible scenario for these effects using a recently constructed three dimensional model of the PS II reaction center.