Loss of inhibition by formate in newly constructed photosystem II D1 mutants, D1-R257E and D1-R257M, of Chlamydomonas reinhardtii

AbstractFormate is known to cause significant inhibition in the electron and proton transfers in photosystem II (PSII); this inhibition is uniquely reversed by bicarbonate. It has been suggested that bicarbonate functions by providing ligands to the non-heme iron and by facilitating protonation of the secondary plastoquinone QB. Numerous lines of evidence indicate an intimate relationship of bicarbonate and formate binding of PSII. To investigate the potential amino acid binding environment of bicarbonate/formate in the QB niche, arginine 257 of the PSII D1 polypeptide in the unicellular green alga Chlamydomonas reinhardtii was mutated into a glutamate (D1-R257E) and a methionine (DQ-R257M). The two mutants share the following characteristics. (1) Both have a drastically reduced sensitivity to formate. (2) A larger fraction of QA− persists after flash illumination, which indicates an altered equilibrium constant of the reaction QA−QB↔QAQB−, in the direction of [QA−], or a larger fraction of non-QB centers. However, there appears to be no significant difference in the rate of electron transfer from QA− to QB. (3) The overall rate of oxygen evolution is significantly reduced, most likely due to changes in the equilibrium constant on the electron acceptor side of PSII or due to a larger fraction in non-QB centers. Additional effects on the donor side cannot yet be excluded. (4) The binding affinity for the herbicide DCMU is unaltered. (5) The mutants grow photosynthetically, but at a decreased (∼70% of the wild type) level. (6) The Fo level was elevated (∼40–50%) which could be due to a decrease in the excitation energy transfer from the antenna to the PSII reaction center, and/or to an increased level of [QA−] in the dark. (7) A decreased (∼10%) ratio of F685 (mainly from CP43) and F695 (mainly from CP47) to F715 (mainly from PSI) emission bands at 77 K suggests a change in the antenna complex. Taken together these results lead to the conclusion that D1-R257 with the positively charged side chain is important for the fully normal functioning of PSII and of growth, and is specially critical for the in vivo binding of formate. Several alternatives are discussed to explain the almost normal functioning of the D1-R257E and D1-R257M mutants