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 mutants 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 increased rate of Q(A)(-) reoxidation. Two strains showed an increase in the amplitude of constant fluorescence (F(o)). Examination of the primary sequence of the combinatorial strains combined with results obtained from analysis of site-directed mutants suggested that alterations in residue 191 of D2 increased the rate of charge recombination. Indeed, reintroduction of Trp191, the residue present in wild type, slowed the Q(A)(-) reoxidation rate in the presence of DCMU by 2-3-fold. However, the nature of other residues, in particular at codon 192, was also important in determining charge recombination rates. The increase in F(o) yield was due to an increased fluorescence lifetime of open reaction centers in intact cells and may reflect a decreased excitation trapping rate in the reaction center. This change was reversed by reintroduction of Trp191 even though a mutant lacking just Trp191 was normal in this respect. Trapping efficiency therefore was decreased only when multiple changes were present at the same time. We interpret Trp191 and neighboring residues to influence the midpoint redox potential of P680/P680(+) and in certain sequence contexts to affect the energy trapping efficiency by P680. The stability or environment of Y(D)(ox) was essentially unaffected in the mutants. Interestingly, many combinatorial mutants displayed an increased requirement for chloride for photoautotrophic growth, and two mutants, C8-10 and C8-23, also required more calcium. This indicates that this CD loop region of D2 not only affects properties of P680 but also affects properties of the oxygen-evolving complex.