Gaozhong Shen

Electrons generated by photosystem II are utilized by an oxidase in the absence of photosystem I in the cyanobacterium Synechocystis sp. PCC 6803

The reduction and reoxidation kinetics of the first quinone‐type electron acceptor in photosystem II, QA −, were measured by fluorescence in a light‐tolerant, photosystem I‐less strain of the cyanobacterium Synechocystis sp. PCC 6803. In this strain, which shows excellent amplitudes of variable fluorescence, the rate of QA − oxidation after photoreduction of the plastoquinone pool was about half of that in the presence of photosystem I. However, upon addition of 5 mM KCN, QA − decay was very slow, and the rate was comparable to that seen in the presence of diuron, which blocks electron transport between QA and QB. The KCN‐imposed block of QA − oxidation was removed efficiently by addition of exogenous quinones that can oxidize the plastoquinone pool. These results indicate that, in the absence of photosystem I, photosystem II‐generated electrons are used very effectively by an oxidase located in the thylakoid; this oxidase may be a component of the respiratory chain.

Q a Binding to D2 Contributes to the Functional and Structural Integrity of Photosystem II

Two D2 mutants were created with a site-directed mutation near the presumable binding site of QA. In one of the mutants, in which Trp-253, the aromatic residue potentially involved in facilitating electron transport from pheophytin to QA and/or in binding of Q A, had been replaced by Leu, PS II was undetectable in thylakoids. This mutant is an obligate photoheterotroph. In another mutant the Gly-215 residue, located next to the His residue that is proposed to bind QA and Fe2+, was mutated to Trp. This mutation leads to a rapid inactivation of oxygen evolution capacity in the light, and to a virtual elimination of the potential to grow photoautotrophically, but does not greatly affect the number of photosystem II reaction centers on a chlorophyll basis. We propose that proper binding of QA to the photosystem II reaction center complex is a prerequisite for stability of the photosystem II complex. Impairment of Q a binding leads to rapid inactivation of photosystem II, which may be followed by a structural disintegration of the complex.

Chimaeric CP47 mutants of the cyanobacterium Synechocystis sp. PCC 6803 carrying spinach sequences: Construction and function.

Chimaeric mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated carrying part or all of the spinach psbB gene, encoding CP47 (one of the chlorophyll-binding core antenna proteins in Photosystem II). The mutant in which the entire psbB gene had been replaced by the homologous gene from spinach was an obligate photoheterotroph and lacked Photosystem II complexes in its thylakoid membranes. However, this strain could be transformed with plasmids carrying selected regions of Synechocystis psbB to give rise to photoautotrophs with a chimaeric spinach/cyanobacterial CP47 protein. This process involved heterologous recombination in the cyanobacterium between psbB sequences from spinach and Synechocystis 6803; which was found to be reasonably effective in Synechocystis. Also other approaches were used that can produce a broad spectrum of chimaeric mutants in a single experiment. Functional characterization of the chimaeric photoautotrophic mutants indicated that if a decrease in the photoautotrophic growth rates was observed, this was correlated with a decrease in the number of Photosystem II reaction centers (on a chlorophyll basis) in the thylakoid membrane and with a decrease in oxygen evolution rates. Remaining Photosystem II reaction centers in these chimaeric mutants appeared to function rather normally, but thermoluminescence and chlorophyll a fluorescence measurements provided evidence for a destabilization of QB−. This illustrates the sensitivity of the functional properties of the PS II reaction center to mild perturbations in a neighboring protein.

Specific mutagenesis of a chlorophyll-binding protein. Progress report.

During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.