Isolation of a highly active photosystem II preparation from Synechocystis 6803 using a histidine-tagged mutant of CP 47.
Site-directed mutagenesis was used to produce a Synechocystis mutant containing a histidine tag at the C terminus of the CP 47 protein of Photosystem II. This mutant cell line, designated HT-3, exhibited slightly above normal rates of oxygen evolution and appeared to accumulate somewhat more Photosystem II reaction centers than a control strain. A rapidly isolatable (<7 h) oxygen-evolving Photosystem II preparation was prepared from HT-3 using dodecyl-beta-d-maltoside solubilization and Co2+ metal affinity chromatography. This histidine-tagged Photosystem II preparation stably evolved oxygen at a high rate (2440 micromol O2 (mg chl)-1 h-1), exhibited an alpha-band absorption maximum at 674 nm, and was highly enriched in a number of Photosystem II components including cytochrome c550. Fluorescence yield analysis using water or hydroxylamine as an electron donor to the Photosystem II preparation indicated that virtually all of the Photosystem II reaction centers were capable of evolving oxygen. Proteins associated with Photosystem II were highly enriched in this preparation. 3,3',5, 5'-Tetramethylbenzidine staining indicated that the histidine-tagged preparation was enriched in cytochromes c550 and b559 and depleted of cytochrome f. This result was confirmed by optical difference spectroscopy. This histidine-tagged Photosystem II preparation may be very useful for the isolation of Photosystem II preparations from mutants containing lesions in other Photosystem II proteins.
Bricker
T M
TM
Department of Biological Sciences, Biochemistry and Molecular Biology Section, Louisiana State University, Baton Rouge, LA 70803, USA. btbric@lsuvm.sncc.lsu.edu
Morvant
J
J
Masri
N
N
Sutton
H M
HM
Frankel
L K
LK
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D001560:Benzidines / Q000378:metabolism; D002846:Chromatography, Affinity; D000458:Cyanobacteria / Q000235:genetics*; D003580:Cytochromes / Q000737:chemistry; D005453:Fluorescence; D006639:Histidine / Q000737:chemistry; D045342:Light-Harvesting Protein Complexes; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010100:Oxygen / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics* / Q000302:isolation & purification*; D045332:Photosystem II Protein Complex; D013053:Spectrophotometry; D014170:Transformation, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D001560:Benzidines; D003580:Cytochromes; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; C070970:photosystem II, chlorophyll-binding protein, CP-47; C020515:N,N,N',N'-tetramethylbenzidine; D006639:Histidine; D010100:Oxygen