Site-Directed Mutagenesis of the CP47 Protein of Photosystem II: Alteration of the Basic Residue 448R to 448G Prevents the Assembly of Functional Photosystem II Centers under Chloride-Limiting Conditions
Abstract
The psbB gene encodes the intrinsic chlorophyll protein CP47 (CPa-1), a component of photosystem II in higher plants, algae, and cyanobacteria. Olignucleotide-directed mutagenesis has been used to introduce mutations into a segment of the psbB gene which encodes the large extrinsic loop E of CP47 in the cyanobacterium Synechocystis sp. PCC 6803. One mutation, R448G, produced a strain with impaired photosystem II activity. When grown in standard BG-11 media (480 µM chloride), this strain grew photoautotrophically at about 50% the rate of control strains and exhibited 63% of the control photosystem II activity. Quantum yield measurement at low light intensities indicated that this mutant had 55% of the fully functional photosystem II centers contained in control strains of Synechocystis. Upon exposure to high light intensities, the mutant strain exhibited a 2.2-fold increase in the rate of photoinactivation. When grown in BG-11 which was depleted in chloride (20 µM chloride), the mutant strain exhibited dramatically altered characteristics. Little or no growth was observed in the mutant while the control strains grew at nearly normal rates. Growth rates of the mutant strain could be restored by the addition of 480 µM bromide to the chloride-deficient BG-11 media. In the presence of glucose, the mutant and control strains grew at comparable rates under either chloride-sufficient or chloride-limiting conditions. Analysis of the mutant cell line grown in the absence of chloride and in the presence of glucose indicated that it exhibited essentially no capacity for oxygen evolution. [14C] Atrazine binding experiments indicated that the mutant assembled 7 5% fewer photosystem II centers than it is capable of assembling in the presence of chloride. Immunological analysis of a number of photosystem II proteins in this mutant indicated that CP43 and the 33-kDa extrinsic manganese-stabilizing protein were present in normal quantities but that CP47 and D1 were present in significantly lower amounts. These results indicate that the mutation R448G in the CP47 protein affects photosystem II assembly and/or stability under chloride-limiting conditions. This is the first mutant identified in any system which exhibits an altered chloride requirement for photosystem II. © 1994, American Chemical Society. All rights reserved.