Oxygen-evolving photosystem II preparation from wild type and photosystem II mutants of Synechocystis sp. PCC 6803.
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.
Kirilovsky
D L
DL
Département de Biologie, URA CNRS 1290, Gif-sur-Yvette, France.
Boussac
A G
AG
van Mieghem
F J
FJ
Ducruet
J M
JM
Sétif
P R
PR
Yu
J J
JJ
Vermaas
W F
WF
Rutherford
A W
AW
D002734:Chlorophyll / Q000378:metabolism; D000077194:Chlorophyll A; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D005798:Genes, Bacterial; D007425:Intracellular Membranes / Q000378:metabolism; D008163:Luminescent Measurements; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*
D045322:Photosynthetic Reaction Center Complex Proteins; D002734:Chlorophyll; D010100:Oxygen; D000077194:Chlorophyll A