In vitro random mutagenesis of the D1 protein of the photosystem II reaction center confers phototolerance on the cyanobacterium Synechocystis sp PCC6803

The D1 protein of the photosystem II reaction center is thought to be the m ost light-sensitive component of the photosynthetic machinery. To understan d the mechanisms underlying the light sensitivity of D1, we performed in vi tro random mutagenesis of the psbA gene that codes for D1, transformed the unicellular cyanobacterium Synechocystis sp, PCC 6803 with mutated psbA, an d selected phototolerant transformants that did not bleach in high intensit y light. A region of psbA2 coding for 178 amino acids of the carboxyl-termi nal portion of the peptide was subjected to random mutagenesis by low fidel ity polymerase chain reaction amplification or by hydroxylamine treatment, This region contains the binding sites for Q(B), D2 (through Fe), and P680, Eighteen phototolerant mutants with single and multiple amino acid substit utions were selected from a half million transformants exposed to white lig ht at 320 mu mol m(-2) s(-1). A strain transformed with non-mutagenized psb A2 became bleached under the same conditions. Site-directed mutagenesis has confirmed that one or more substitutions of amino acids at residues 234, 2 54, 260, 267, 322, 326, and 328 confers phototolerance, The rate of degrada tion of D1 protein was not appreciably affected by the mutations. Reduced b leaching of mutant cyanobacterial cells may result from continued buildup o f photosynthetic pigment systems caused by changes in redox signals origina ting from D1.