Highly purified thermo-stable oxygen-evolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43.
The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex. The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni(2+)-affinity column chromatography. The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 mumol (mg Chl)-1 h-1 at 45 degrees C with ferricyanide as an electron acceptor. The core complex emitted thermoluminescence B2-, B1- and Q-bands arising from S2QB-, S3QB- and S2QA- charge recombinations at respective emission temperatures of 45, 38 and 20 degrees C, all of which were higher by about 15 degrees C as compared with those in mesophilic spinach BBY membranes. These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs. The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20 degrees C for 8 d. These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII.
Sugiura
M
M
Photosynthesis Research Laboratory, RIKEN (The Institute of Physical and Chemical Research, Saitama, Japan.
Inoue
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D019281:Dimerization; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006639:Histidine / Q000737:chemistry; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D045332:Photosystem II Protein Complex; D020836:Protein Structure, Quaternary; D013053:Spectrophotometry; D013696:Temperature
D017931:DNA Primers; D004269:DNA, Bacterial; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; C083100:photosystem II, chlorophyll binding protein, CP-43; D006639:Histidine; D010100:Oxygen