SITE-DIRECTED MUTAGENESIS OF THE CP47 PROTEIN OF PHOTOSYSTEM-II - ALTERATION OF THE BASIC RESIDUE (448)R TO (448)G PREVENTS THE ASSEMBLY OFFUNCTIONAL PHOTOSYSTEM-II CENTERS UNDER CHLORIDE-LIMITING CONDITIONS

The psbB gene encodes the intrinsic chlorophyll protein CP47 (CPa-1), a component of photosystem II in higher plants, algae, and cyanobacter ia. Olignucleotide-directed mutagenesis has been used to introduce mut ations into a segment of the psbB gene which encodes the large extrins ic loop E of CP47 in the cyanobacterium Synechocystis sp. PCC 6803. On e mutation, R448G, produced a strain with impaired photosystem II acti vity. When grown in standard BG-11 media (480 mu M chloride), this str ain grew photoautotrophically at about 50% the rate of control strains and exhibited 63% of the control photosystem II activity. Quantum yie ld measurement at low light intensities indicated that this mutant had 55% of the fully functional photosystem II centers contained in contr ol strains of Synechocystis. Upon exposure to high light intensities, the mutant strain exhibited a 2.2-fold increase in the rate of photoin activation. When grown in BG-11 which was depleted in chloride (20 mu M chloride), the mutant strain exhibited dramatically altered characte ristics. Little or no growth was observed in the mutant while the cont rol strains grew at nearly normal rates. Growth rates of the mutant st rain could be restored by the addition of 480 mu M bromide to the chlo ride-deficient BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates under either chloride-suffic ient or chloride-limiting conditions. Analysis of the mutant cell line grown in the absence of chloride and in the presence of glucose indic ated that it exhibited essentially no capacity for oxygen evolution. [ C-14]Atrazine binding experiments indicated that the mutant assembled 75% fewer photosystem II centers than it is capable of assembling in t he presence of chloride. Immunological analysis of a number of photosy stem II proteins in this mutant indicated that CP43 and the 33-kDa ext rinsic manganese-stabilizing protein were present in normal quantities but that CP47 and D1 were present in significantly lower amounts. The se results indicate that the mutation R448G in the CP47 protein affect s photosystem II assembly and/or stability under chloride-limiting con ditions. This is the first mutant identified in any system which exhib its an altered chloride requirement for photosystem II.