Functional analysis of combinatorial mutants with changes in the C-terminus of the CD loop of the D2 protein in photosystem II of Synechocystis sp PCC 6803

Photosystem II properties were investigated in a set of combinatorial mutan ts containing changes in the C-terminal end of the CD lumenal loop (Gly187- Asn194) in the D2 protein of Synechocystis sp. PCC 6803. Initial screening of variable fluorescence (F-v) induction and decay in the presence of DCMU showed that all but one of the combinatorial strains tested had an increase d rate of QA(-) reoxidation. Two strains showed an increase in the amplitud e of constant fluorescence (F-o). Examination of the primary sequence of th e combinatorial strains combined with results obtained from analysis of sit e-directed mutants suggested that alterations in residue 191 of D2 increase d the rate of charge recombination. Indeed, reintroduction of Trp 191, the residue present in wild type, slowed the Q(A)- reoxidation rate in the pres ence of DCMU by 2-3-fold. However, the nature of other residues, in particu lar at codon 192, was also important in determining charge recombination ra tes. The increase in F-o yield was due to an increased fluorescence lifetim e of open reaction centers in intact cells and may reflect a decreased exci tation trapping rate in the reaction center. This change was reversed by re introduction of Trp191 even though a mutant lacking just Trp191 was normal in this respect. Trapping efficiency therefore was decreased only when mult iple changes were present at the same time. We interpret Trp191 and neighbo ring residues to influence the midpoint redox potential of P680/P680(+) and in certain sequence contexts to affect the energy trapping efficiency by P 680. The stability or environment of Y-D(ox) was essentially unaffected in the mutants. Interestingly, many combinatorial mutants displayed an increas ed requirement for chloride for photoautotrophic growth, and two mutants, C 8-10 and C8-23, also required more calcium. This indicates that this CD loo p region of D2 not only affects properties of P680 but also affects propert ies of the oxygen-evolving complex.