The cysteine-proximal aspartates in the F(x)-binding niche of photosystem. I: Effect of alanine and lysine replacements on photoautotrophic growth, electron transfer rates, single-turnover flash efficiency, and EPR spectral properties

The F(x) electron acceptor in Photosystem I (PS I) is a highly electronegative (E(m) = -705 mV) interpolypeptide [4Fe-4S] cluster ligated by cysteines 556 and 565 on PsaB and cysteines 574 and 583 on PsaA in Synechocystis sp. PCC 6803. An aspartic acid is adjacent to each of these cysteines on PsaB and adjacent to the proline-proximal cysteine on PsaA. We investigated the effect of D566(PsaB) and D557(PsaB) on electron transfer through F(x) by changing each aspartate to the neutral alanine or to the positively charged lysine either singly (D566A(PsaB), D557A(PsaB), D566K(PsaB), and D557K(PsaB)) or in pairs (D557A(PsaB)/D566A(PsaB) and D557K(PsaB)/D566A(PsaB)). All mutants except for D557K(PsaB)/D566A(PsaB) grew photoautotrophically, but the growth of D557K(PsaB) and D557A(PsaB)/D566A(PsaB) was impaired under low light. The doubling time was increased, and the chlorophyll content per cell was lower in D557K(PsaB) and D557A(PsaB)/D566A(PsaB) relative to the wild type and the other mutants. Nevertheless, the rates of NADP⁺ photoreduction in PSI complexes from all mutants were no less than 75% of that of the wild type. The kinetics of back- reaction of the electron acceptors on a single-turnover flash showed efficient electron transfer to the terminal acceptors F(A) and F(B) in PSI complexes from all mutants. The EPR spectrum of F(x) was identical to that in the wild type in all but the single and double D566A(PsaB) mutants, where the high-field resonance was shifted downfield. We conclude that the impaired growth of some of the mutants is related to a reduced accumulation of PSI rather than to photosynthetic efficiency. The chemical nature and the charge of the amino acids adjacent to the cysteine ligands on PsaB do not appear to be significant factors in the efficiency of electron transfer through F(x).