Construction and characterization of a photosystem II D1 mutant (arginine-269-glycine) of Chlamydomonas reinhardtii.
Numerous lines of evidence indicate that bicarbonate anion regulates electron and proton transfer processes in the photosystem II (PSII) complex of chloroplasts and cyanobacteria. On the reducing side of PSII, the addition of bicarbonate to bicarbonate-depleted (or formate-treated) membranes accelerates, especially, QA(-)-->QB(-) electron transfer kinetics. The site(s) at which bicarbonate binds is unknown. It is evident, however, from several spectroscopic studies that the bicarbonate binding site on the reducing side of PSII includes the non-heme iron located between the QA and QB sites. Since small anions may displace bicarbonate (Good, N.E. (1963) Plant Physiol. 38, 298-304) [1], it is apparent that the bicarbonate binding site is electrostatic in nature, presumably also involving positively charged amino acid residues. Previously, it had been predicted that residue arginine 269 of the PSII D1 protein may participate in bicarbonate binding. To test this hypothesis, we have generated a non-conservative mutation in the psbA gene of Chlamydomonas reinhardtii which converts residue R269 to a glycine (R269G). The R269G mutant was unable to grow photosynthetically or evolve oxygen. This phenotype is associated with a lack of the tetra-manganese water splitting complex and a reduced capacity to form a stabilized charge separated state (defined as TyrD+/QA- under the experimental conditions measured). In addition, the mutant cells have a less stable PSII complex than wild-type cells, particularly when grown in the light. It is apparent from analyses of the effect of formate on the magnitude of the QA-Fe+2 EPR signal, however, that the bicarbonate or formate binding site is not substantially affected by the R269G mutation. Although our results do not substantiate that residue R269 is the site at which bicarbonate is bound, they demonstrate the importance of R269 in the structure and function of PSII. It is apparent from analysis of the photosynthetic phenotype, that the structural perturbations on the stromal side of the D1 protein are transduced to the lumenal side of the membrane altering charge accumulating processes on the electron donor side of PSII.
Hutchison
R S
RS
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
Xiong
J
J
Sayre
R T
RT
Govindjee
D000818:Animals; D001120:Arginine / Q000235:genetics*; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D005998:Glycine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D045332:Photosystem II Protein Complex
D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D001120:Arginine; D005998:Glycine