Towards structural determination of the water-splitting enzyme. Purification, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium.
A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the Q(B) binding site. The oxygen-evolving activity, 5000-6000 micromol of O(2)/(h.mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50 degrees C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 A. The space group was determined to be P2(1)2(1)2(1) with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization.
Kuhl
H
H
Department of Plant Biochemistry, Faculty of Biology, Ruhr-University Bochum, D-44780 Bochum, Germany.
Kruip
J
J
Seidler
A
A
Krieger-Liszkay
A
A
Bunker
M
M
Bald
D
D
Scheidig
A J
AJ
Rögner
M
M
D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000737:chemistry*; D019281:Dimerization; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D046911:Macromolecular Substances; D008958:Models, Molecular; D010100:Oxygen / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013053:Spectrophotometry; D013696:Temperature; D014867:Water / Q000378:metabolism*
D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D014867:Water; D010100:Oxygen