Biochemical and spectroscopic characterization of a new oxygen-evolving photosystem II core complex from the cyanobacterium Synechocystis PCC 6803.
We describe here a new procedure permitting rapid (12-13 h) isolation of a pure oxygen-evolving photosystem II (PSII) core complex from the cyanobacterium Synechocystis PCC 6803. This procedure involves dodecyl maltoside extraction of thylakoid membranes followed by single-step column chromatography using a weak anion-exchanger. SDS-PAGE and immunoblotting show that the complex consists of five intrinsic membrane proteins (CP47, CP43, D1, D1, and cyt b559), one extrinsic protein (MSP), and one unknown protein with a molecular mass of approximately 26 kDa. A chemical and functional analysis, normalized to 2 molecules of pheophytin a, indicates that this PSII core complex contains 1 photoactive plastoquinone, QA, 4 manganese atoms, 38 chlorophyll a molecules, 1 cytochrome b559, 2 plastoquinone-9, and 9-10 beta-carotenes. The complex exhibits high rates of oxygen evolution, typically 2400-2600 mumol of O2 (mg of Chl)-1 h-1 in the presence of 2,5-dichlorobenzoquinone as an artificial electron acceptor with a pH optimum of 6.5. A strong light minus dark multiline EPR signal, arising from the S2 state of the oxygen-evolving complex (OEC), is observed at 10 K following illumination at 198 K. The determination of the absolute oxygen yield per saturating microsecond flash indicates that essentially all of the PSII centers contain functional oxygen-evolving complexes. This point is further supported by the absence of photoaccumulation, upon room temperature illumination, of the immediate oxidant of the OEC, redox-active tyrosine, YZ.. On the basis of EPR spectra, oxidized minus reduced difference spectra, and SDS-PAGE, the preparation contains on a per mole basis with PSII only trace amounts of PSI (approximately 0.04), cytochrome b6/f complex (< or = 0.01), and ATPase (< or = 0.05). All of these results indicate that this PSII preparation is to date the most highly purified oxygen-evolving core complex from Synechocystis 6803 that retains all of the reaction centers active for oxygen evolution. As Synechocystis 6803 is being used extensively for site-directed mutagenesis of PSII, this preparation is particularly valuable for spectroscopic and biochemical analyses of PSII from wild-type and from site-directed mutants.
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Central Research and Development Department, E. I. Du Pont de Nemours & Company, Wilmington, Delaware 19880-0173.
Diner
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D002118:Calcium / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D000458:Cyanobacteria / Q000737:chemistry*; D003573:Cytochrome b Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D045342:Light-Harvesting Protein Complexes; D015636:Magnesium Chloride / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D045332:Photosystem II Protein Complex; D012965:Sodium Chloride / Q000494:pharmacology; D013053:Spectrophotometry
D003573:Cytochrome b Group; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D015636:Magnesium Chloride; D012965:Sodium Chloride; C019313:cytochrome b559; D002122:Calcium Chloride; D010100:Oxygen; D002118:Calcium