Oxygen-evolving photosystem II preparation from wild type and photosystem II mutants of Synechocystis sp. PCC 6803

• Published in: Biochemistry (Easton) 1992-02, Vol.31 (7), p.2099-2107
• Main Authors: Kirilovsky, D L, Boussac, A G, van Mieghem, F J, Ducruet, J M, Sétif, P R, Yu, J J, Vermaas, W F, Rutherford, A W
• Format: Article
• Language: English
• Subjects: 140505 - Solar Energy Conversion- Photochemical, Photobiological, & Thermochemical Conversion- (1980-); chemical analysis; Chlorophyll - metabolism; Chlorophyll A; CYANOBACTERIA; Cyanobacteria - genetics; Cyanobacteria - metabolism; ELECTRON SPIN RESONANCE; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; ELEMENTS; EMISSION SPECTRA; enzymes; Fluorescence Polarization; Genes, Bacterial; incorporation; Intracellular Membranes - metabolism; Luminescent Measurements; MAGNETIC RESONANCE; MEMBRANE PROTEINS; MEMBRANES; methodology; MICROORGANISMS; MUTANTS; Mutation; NONMETALS; ORGANIC COMPOUNDS; OXYGEN; Oxygen - metabolism; particles; photochemical reactions; photosynthesis; photosynthetic apparatus; PHOTOSYNTHETIC MEMBRANES; Photosynthetic Reaction Center Complex Proteins - metabolism; PHOTOSYNTHETIC REACTION CENTERS; photosystem II; PROTEINS; PURIFICATION; RESONANCE; SOLAR ENERGY; SPECTRA; Synechocystis; THYLAKOID MEMBRANE PROTEINS
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00122a030

We present here a simple and rapid method which allows relatively large quantities of oxygen-evolving photosystem II- (PS-II-) enriched particles to be obtained from wild-type and mutants of the cyanobacterium Synechocystis 6803. This method is based on that of Burnap et al. [Burnap, R., Koike, H., Sotiropoulou, G., Sherman, L. A., & Inoue, Y. (1989) Photosynth. Res. 22, 123-130] but is modified so that the whole preparation, from cells to PS-II particles, is achieved in 10 h and involves only one purification step. The purified preparation exhibits a 5-6-fold increase of O2-evolution activity on a chlorophyll basis over the thylakoids. The ratio of PS-I to PS-II is about 0.14:1 in the preparation. The secondary quinone electron acceptor, QB, is present in this preparation as demonstrated by thermoluminescence studies. These PS-II particles are well-suited to spectroscopic studies as demonstrated by the range of EPR signals arising from components of PS-II that are easily detectable. Among the EPR signals presented are those from a formal S3-state, attributed to an oxidized amino acid interacting magnetically with the Mn complex in Ca(2+)-deficient PS-II particles, and from S2 modified by the replacement of Ca2+ by Sr2+. Neither of these signals has been previously reported in cyanobacteria. Their detection under these conditions indicates a similar lesion caused by Ca2+ depletion in both plants and cyanobacteria. The protocol has also been applied to mutants which have site-specific changes in PS-II. Data are presented on mutants having changes on the electron donor (Y160F) and electron acceptor (G215W) side of the D2 polypeptide.