Epitope mapping of the monoclonal antibody FAC2 on the apoprotein of CPa-1 in photosystem II

Using a combination of cyanogen bromide cleavage and endoproteinase digestion we have shown that the putative epitope for the monoclonal antibody FAC2 lies in the region 360Pro-391Ser on the apoprotein of CPa-1. This region lies entirely within the large extrinsic loop of this protein. We have shown previously that the epitope of FAC2 becomes exposed in oxygen-evolving membranes upon treatment with alkaline Tris which releases all four of the manganese associated with the oxygen-evolving site of photosystem II. The epitope is not exposed, however, after CaCl2 treatment and exposure to low concentrations of chloride, conditions which lead to the release of two of the four manganeses associated with the oxygen-evolving site [(1987) Arch. Biochem. Biophys. 256, 295-301]. These results suggest that, upon release of the chloride-insensitive manganese from photosystem II membranes, a conformational change occurs which leads to the exposure of 360Pro-391Ser on CPa-1 to the monoclonal antibody FAC2. © 1989

Carboxylate groups on the manganese-stabilizing protein are required for efficient binding of the 24 kDa extrinsic protein to photosystem II

The effects of the modification of carboxylate groups on the manganese-stabilizing protein on the binding of the 24 kDa extrinsic protein to Photosystem II were investigated. Carboxylate groups on the manganese-stabilizing protein were modified with glycine methyl ester in a reaction facilitated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The manganese-stabilizing protein which was modified while associated with NaCl-washed membranes could bind to calcium chloride-washed PS II membranes and reconstitute oxygen evolution in a manner similar to that observed for unmodified manganese-stabilizing protein (Frankel, L,K, Cruz, J. C. and Bricker, T. M. (1999) Biochemistry 38, 14271-14278), However, PS II membranes reconstituted with this modified protein were defective in their ability to bind the extrinsic 24 kDa protein of Photosystem II. Mapping of the sites of modification was carried out by trypsin and Staphylococcus V8 protease digestion of the modified protein and analysis by MALDI mass spectrometry. These studies indicated that the domains 1E-71D, 97D-144D, and 180D-187E are labeled when the manganesestabilizing protein is bound to NaCl-washed Photosystem II membranes. We hypothesize that modified carboxylates, possibly residues 1E, 32E, 139E, and/or 187E, in these domains are responsible for the altered binding affinity of the 24 kDa protein observed

The psbo1 mutant of Arabidopsis cannot efficiently use calcium in support of oxygen evolution by photosystem II

The Arabidopsis thaliana mutant psbo1 contains a point mutation in the psbO-1 gene (At5g66570) leading to the loss of expression of the PsbO-1 protein and overexpression of the PsbO-2 protein (Murakami, R., Ifuku, K., Takabayashi, A., Shikanai, T., Endo, T., and Sato, F. (2002) FEBS Lett. 523, 138-142). Previous characterization of fluorescence induction and decay kinetics by our laboratory documented defects on both the oxidizing and reducing sides of Photosystem II. Additionally, anomalous flash oxygen yield patterns indicated that the mutant contains a defective oxygen-evolving complex that appears to exhibit anomalously long-lived S2 and S3 oxidation states (Liu, H., Frankel, L. K., and Bricker, T. M. (2007) Biochemistry 46, 7607-7613). In this study, we have documented that the S2 and S3 states in psbo1 thylakoids decay very slowly. The total flash oxygen yield of the psbo1 mutant was also significantly reduced, as was its stability. Incubation of psbo1 thylakoids at high NaCl concentrations did not increase the rate of S2 and S3 state decay. The oxygen-evolving complexes of the mutant did, however, exhibit somewhat enhanced stability following this treatment. Incubation with CaCl2 had a significantly more dramatic effect. Under this condition, both the S2 and S 3 states of the mutant decayed at nearly the same rate as the wild type, and the total oxygen yield and its stability following CaCl2 treatment were indistinguishable from that of the wild type. These results strongly suggest that the principal defect in the psbo1 mutant is an inability to effectively utilize the calcium associated with Photosystem II. We hypothesize that the PsbO-2 protein cannot effectively sequester calcium at the oxygen-evolving site. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc

Functional complementation of the Arabidopsis thaliana psbo1 mutant phenotype with an N-terminally His\u3csub\u3e6\u3c/sub\u3e-tagged PsbO-1 protein in photosystem II

The Arabidopsis thaliana mutant psbo1 has recently been described and characterized. Loss of expression of the PsbO-1 protein leads to a variety of functional perturbations including elevated levels of the PsbO-2 protein and defects on both the oxidizing- and reducing-sides of Photosystem II. In this communication, two plant lines were produced using the psbo1 mutant as transgenic host, which contained an N-terminally histidine6-tagged PsbO-1 protein. This protein was expressed and correctly targeted into the thylakoid lumen. Immunological analysis indicated that different levels of expression of the modified PsbO-1 protein were obtained in different transgenic plant lines and that the level of expression in each line was stable over several generations. Examination of the Photosystem II closure kinetics demonstrated that the defective double reduction of QB and the delayed exchange of QBH2 with the plastoquinone pool which were observed during the characterization of the psbo1 mutant were effectively restored to wild-type levels by the His6-tagged PsbO-1 protein. Flash fluorescence induction and decay were also examined. Our results indicated that high expression of the modified PsbO-1 was required to increase the ratio of PS IIα/PS IIβ reaction centers to wild-type levels. Fluorescence decay kinetics in the absence of DCMU indicated that the expression of the His6-tagged PsbO-1 protein restored efficient electron transfer to QB, while in the presence of DCMU, charge recombination between QA- and the S2 state of the oxygen-evolving complex occurred at near wild-type rates. Our results indicate that high expression of the His6-tagged PsbO-1 protein efficiently complements nearly all of the photochemical defects observed in the psbo1 mutant. Additionally, this study establishes a platform on which the in vivo consequences of site-directed mutagenesis of the PsbO-1 protein can be examined. © 2009 Elsevier B.V. All rights reserved

Effects of chloride on paramagnetic coupling of manganese in calcium chloride-washed photosystem II preparations

The effect of chloride on paramagnetic coupling of manganese in the oxygen-evolving complex of CaCl2--washed PS II preparations was examined using Q-band ESR. When these PS II preparations were depleted of chloride, a strong 6-line ESR signal characteristic of protein-bound, uncoupled manganese was observed. Incubation at high chloride concentrations caused the disappearance of this signal. By repeated removal and addition of chloride, the signal could be cycled on and off without loss of bound manganese. When in a chloride-depleted state, the ESR-detectable protein-bound manganese could be removed by treatment with EDTA. Subsequent heating of EDTA-treated preparations revealed a second pool of protein-bound manganese associated with PS II. One of these pools requires a high concentration of chloride to maintain paramagnetic coupling while the second pool (within the limits of our observations) does not appear to require chloride for the maintenance of the paramagnetically coupled state. © 1986

Kinetic characterization of His-tagged CP47 Photosystem II in Synechocystis sp. PCC6803

Recently, construction of strains of Synechocystis sp. PCC6803 having a His6 extension (His-tag) of the carboxyl terminus of the CP47 protein has been reported (T.M. Bricker et al, Biochim. Biophys. Acta 1409 (1998) 50; M.J. Reifler et al., in: Garab, Pusztai (Eds.) Proc. XIth International Congress on Photosynthesis, 1998). While these initial reports suggest a minimal impact of the His-tag upon Photosystem (PS) II function, a more thorough analysis of the kinetic properties of the modified complex is essential. This communication reports on a more detailed kinetic analysis to assess possible perturbations of PS II due to the genetic addition of the His-tag on the CP47 protein. It was found that: (1) Patterns of flash O2 yield exhibited normal period four oscillations and the associated fits of the Kok-Joliot S-state cycling parameters were virtually identical to the wild type; (2) O2 release kinetics during the S3-S0 transition were experimentally indistinguishable from the wild type; (3) S-state decay measurements indicate slightly faster decays of the S2 and S3 states compared to the wild type; (4) fluorescence measurements indicate that the kinetics of the forward reaction of electron transfer from Q(A)/- to Q(B) and back-reactions of Q(A)/- with PS II electron donors are similar in the His-tag and wild-type strains. It is therefore concluded that the addition of the His-tag results in a minimal perturbation of PS II function. (C) 2000 Elsevier Science B.V