Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers?

Abstract

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, Q A and Q B. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g. Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides, RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered Glu M234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P +) in the absence of exogenous donor, i.e., the recombination of P +Q A − or P +Q B −, respectively; (b) the kinetics of electron transfer from Q A − to Q B; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P + (Q B turnover). The absence of a formate-bicarbonate effect in these mutants suggests that Glu M234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of Q A. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (τ = 3.5ps), followed by reduction of the bacteriopheophytin (τ = 0.9ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of Q A was slowed by a factor of 2 (τ = 410 ± 30and47 ± 30ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type). EPR studies of the isolated RCs showed a characteristic g = 1.82 signal for the Q A semiquinone coupled to the iron atom, which was indistinguishable from the wild type. It is concluded that Glu M234 is not essential to the normal functioning of the acceptor quinone complex in bacterial RCs and that the role of bicarbonate in PS II is distinct from the role of this residue in bacterial RCs.