Site-Directed Photosystem II Mutants with Perturbed Oxygen-Evolving Properties. 1. Instability or Inefficient Assembly of the Manganese Cluster In vivo

Abstract

Several site-directed photosystem II mutants with substitutions at Asp-170 of the D1 polypeptide were characterized by noninvasive methods in vivo. In several mutants, including some that evolve oxygen, a significant fraction of photosystem II reaction centers are shown to lack photooxidizable Mn ions. In this fraction of reaction centers, either the high-affinity site from which Mn ions rapidly reduce the oxidized secondary electron donor, YZ+, is devoid of Mn ions or the Mn ion(s) bound at this site are unable to reduce YZ+. It is concluded that the Mn clusters in these mutants are unstable or are assembled inefficiently in vivo. Mutants were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The in vivo characterization procedures employed in this study involved measuring changes in the yield of variable chlorophyll a fluorescence following a saturating flash or brief illumination given in the presence of the electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, or following each of a series of saturating flashes given in the absence of this inhibitor. These procedures are easily applied to mutants that evolve little or no oxygen, facilitate the characterization of mutants with labile oxygen-evolving complexes, permit photosystem II isolation efforts to be concentrated on mutants having the stablest Mn clusters, and guide systematic spectroscopic studies of isolated photosystem II particles to mutants of particular interest.

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

“…To test the possible role of cyt b 559 under donor-side photoinhibitory conditions, we constructed D170AD1/H22K␣ and D170AD1/Y18S␣ double mutants. D170AD1 mutant cells are able to accumulate stable PSII to the wild-type level but cannot evolve any oxygen (33). Interestingly, our results showed that little PSII was present in D170AD1/ H22K␣ and D170AD1/Y18S␣ double mutants (see Table 1).…”

Glutamate-354 of the CP43 polypeptide interacts with the oxygen-evolving Mn4Ca cluster of photosystem II: a preliminary characterization of the Glu354Gln mutant

“…Large-scale liquid cultures (3!7 l) were propagated as described previously (Strickler et al 2007). To verify the integrity of the mutant cultures that were harvested for the purification of thylakoid membranes and PSII particles, an aliquot of each culture was set aside and the sequence of the portion of the psbC gene that encodes the large extrinsic loop of CP43 was obtained after PCR amplification of genomic DNA (Chu et al 1994). No trace of the wild-type codon was detected in any of the mutant cultures.…”

Protein ligation of the photosynthetic oxygen-evolving center

“…This mutant is weakly photoautotrophic and assembles Mn 4 Ca clusters in ~ 50% of its PSII reaction centers. These assembled Mn 4 Ca clusters function normally [47,52,53] and exhibit normal S 1 and S 2 state multiline EPR signals [50]. In addition, two FTIR studies have shown that the S 2 -minus-S 1 FTIR difference spectrum of D1-Asp170His PSII particles is essentially unchanged from that of wild-type PSII particles [43,46].…”