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More than one hundred mutants of Synechocystis sp. PCC 6803 impaired in photoautotrophic growth were generated by in vitro random PCR mutagenesis targeted to a region of the psbAII gene corresponding to a 210 amino acid (Ser148–Ala357) segment of the D1 protein. The 90 random mutants that could translate the full-length D1 protein carried 1–9 (on average 3.0) amino acid substitutions in the targeted region. Mutations were often found in the obligate photoheterotrophic strains at specific residues that have been reported or speculated to be important in the function of PSII, such as Y161, H198, H272, E333 and H337. This verifies the usefulness of the present method to identify functionally important residues in PSII. Other residues that were often mutated in the strains with impaired photoautotrophy included non-charged residues around the lumenal edges of transmembrane helices C, D and E, such as I192 and N296. Eleven mutants carried a single-point mutation in residues, such as Q165, Q187, W278, A294 and N298, and these identified the functional importance of these residues, most of which were on the donor side of PSII. A preliminary characterization of some of the mutants obtained in this study is provided.

Random mutations were introduced into the DNA fragment of the psbA2 gene of Synechocystis sp. PCC 6803, which encodes the carboxyl-terminal 178 amino acid region of the D1 protein of the PSII reaction center, by in vitro random mutagenesis to obtain D1 species resistant to herbicides and to understand the protein-herbicide interactions. The mutants were screened on the criterion of resistance to either 1 microM DCMU or 10 microM atrazine. In these mutants, amino acid substitutions were distributed throughout the entire area of the targeted region in the D1 protein. However, in every mutant, except for one case, the substitution was present in the region described as the "herbicide-binding niche", i.e., between Phe211 and Leu275, although some amino acid substitutions which were not previously described were found at residues known to be involved with herbicide affinity. Thus, the result of random mutagenesis basically supports the validity of the proposed structural model for the D1 protein, as well as of the herbicide-binding niche. Preliminary characterization of the herbicide-resistant mutants obtained in this study has also been conducted.

Transient absorbance changes of the primary electron donor chlorophyll a (P680) and acceptor pheophytin a (H) were measured at 77 K by nanosecond laser spectroscopy in the Dl-D2-cytochrome bSS9 photosystem II reaction center complex containing dibromomethylisopropyl benzoquinone (DBMIB). After the laser excitation of the reaction center in the presence of DBMIB, only the P680+(DBMIB) state was detected. P680+ mainly decayed with a t 1/e of 11 ms. In the absence of DBMIB, the excitation produced the P680+H~ radical pair. The radical pair produced the triplet state (P680T) with a t 1/e of 50 ns, and P680T then decayed with a tVt of 2.1 ms. It was concluded that H~ was oxidized by DBMIB in a time range faster than the detecting time resolution (3.5 ns) even at 77 K. The rapid oxidation of H~ by DBMIB was also confirmed by the suppression of delayed fluorescence with a decay t1/e of 50 ns. The P680+(DBMIB")/P680(DBMIB) difference spectrum exhibited a Q, band with a peak at 682 nm with a shoulder at 673 nm. The spectral shape was almost temperature insensitive between 77 and 265 K. The feature of this spectrum in the wavelength range between 330 and 720 nm was compared with that of P680 T/P680 or H"/H at 77 K.

The D1 and D2 subunits of the reaction center of photosystem II are intrinsic proteins. A procedure was developed for the separation and purification of these two proteins on a large scale from the photosystem II reaction center complex of spinach by high-performance liquid chromatography on a gel-permeation column. The purified D1 and D2 proteins were subjected to determination of their carboxy-terminal sequences by digestion of the proteins with carboxypeptidase Y