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. Oligonucleotide-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 microM 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 microM 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 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed mutagenesis of the CP 47 protein of photosystem II: 167W in the lumenally exposed loop C is required for photosystem II assembly and stability

“…W167S, however, exhibited a significantly rapid loss in oxygen evolution capacity indicating that its PS II reaction centers are most susceptible to photoinactivation. The increased susceptibility of W167S to photoinactivation appears comparable in magnitude to that observed in the E69Q and P 161L mutants in the D2 protein [25] and the R448G mutant in CP 47 of Synechocystis [22]. The E69Q mutation is believed to affect the stability and/or ligation of the manganese cluster while the P161L mutation is located near YD and is believed to affect the efficiency of transfer of electrons from the oxygen-evolving site to Y+ [25].…”

“…Restriction digests, cloning, growth and transformation of bacterial strains, and isolation of DNA were performed according to standard procedures [17,28]. Synechocystis transformation procedures and the characteristics of control strain K3 have been described elsewhere [21,22]. The desired mutation was substituted into pTZI8K9 by oligonucleotide-directed mutagenesis using the procedure of Kunkel et al [ 16].…”

“…The E69Q mutation is believed to affect the stability and/or ligation of the manganese cluster while the P161L mutation is located near YD and is believed to affect the efficiency of transfer of electrons from the oxygen-evolving site to Y+ [25]. The R448G mutant in CP 47 is believed to alter a chloride-binding site in PS II [22]. These authors hypothesized that the decreased rates of electron transfer from the oxygenevolving complex to Yz + which was observed in these mutants resulted in the accumulation of oxidizing-side radicals (such as Yz + and P680 +) which damage the PS II reaction center and lead to the observed photoinactivation.…”

Mutations in the CP43 Protein of Photosystem II Affect PSII Function and Cytochrome C550 Binding

“…This was the first mutant of this type described in the CP43 protein. In terms of this chloride effect, R320S has a similar phenotype to the R448G, K321G, and F363R mutants constructed in the CP47 large extrinsic loop (Putnam-Evans & Bricker, 1994Clarke & Eaton-Rye, 1999). These results were significant because the R320S mutation represented the first site in the CP43 protein that alters the chloride requirement of PSII and inferred that the large extrinsic loop of CP43 is involved in chloride binding/sequestration at the PSII active site.…”

Mössbauer spectroscopy in studies of photosynthesis

“…The PSII core is additionally formed by two components of the proximal antennae, CP43 and CP47, responsible for transferring sunlight to the reaction center P680 (tetramer of chlorophylls) and cytochrome b 559 (cyt. b 559 ) required for the formation of a functional PSII complex [3][4][5][6][7][8][9][10]. The catalytic site of water oxidation, formed by four manganese ions and one calcium ion [11][12][13] is stabilized by three extrinsic proteins of molecular masses 33 kDa, 24 kDa and 17 kDa in case of higher plants [11,14].…”