Binding and functional properties of the extrinsic proteins in oxygen-evolving photosystem II particle from a green alga, Chlamydomonas reinhardtii having his-tagged CP47.
Oxygen-evolving photosystem II (PSII) particles were purified from Chlamydomonas reinhardtii having His-tag extension at the C terminus of the CP47 protein, by a single-step Ni(2+)-affinity column chromatography after solubilization of thylakoid membranes with sucrose monolaurate. The PSII particles consisted of, in addition to intrinsic proteins, three extrinsic proteins of 33, 23 and 17 kDa. The preparation showed a high oxygen-evolving activity of 2,300-2,500 micro mol O(2) (mg Chl)(-1) h(-1) in the presence of Ca(2+) using ferricyanide as the electron acceptor, while its activity was 680-720 micro mol O(2) (mg Chl)(-1) h(-1) in the absence of Ca(2+) and Cl(-) ions. The activity was 710-820 micro mol O(2) (mg Chl)(-1) h(-1) independent of the presence or absence of Ca(2+) and Cl(-) when 2,6-dichloro-p-benzoquinone was used as the acceptor. These activities were scarcely inhibited by DCMU. The kinetics of flash-induced fluorescence decay revealed that the electron transfer from Q(A)(-) to Q(B) was significantly inhibited, and the electron transfer from Q(A)(-) to ferricyanide was largely stimulated in the presence of Ca(2+). These results indicate that the acceptor side, Q(B) site, was altered in the PSII particles but its donor side remained intact. Release-reconstitution experiments revealed that the extrinsic 23 and 17 kDa proteins were released only partially by NaCl-wash, while most of the three extrinsic proteins were removed when treated with urea/NaCl, alkaline Tris or CaCl(2). The 23 and 17 kDa proteins directly bound to PSII independent of the other extrinsic proteins, and the 33 kDa protein functionally re-bound to CaCl(2)-treated PSII which had been reconstituted with the 23 and 17 kDa proteins. These binding properties were largely different from those of the extrinsic proteins in higher plant PSII, and suggest that each of the three extrinsic proteins has their own binding sites independent of the others in the green algal PSII.
Suzuki
Takehiro
T
Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601 Japan.
Minagawa
Jun
J
Tomo
Tatsuya
T
Sonoike
Kintake
K
Ohta
Hisataka
H
Enami
Isao
I
D020418:Algal Proteins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology; D002846:Chromatography, Affinity; D004237:Diuron / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D005292:Ferricyanides / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism*; D010788:Photosynthesis / Q000502:physiology*; D011485:Protein Binding / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D020524:Thylakoids / Q000187:drug effects / Q000378:metabolism
D020418:Algal Proteins; D002712:Chlorides; D005292:Ferricyanides; C007931:hexacyanoferrate III; D012965:Sodium Chloride; D004237:Diuron; D008274:Magnesium; D002122:Calcium Chloride; D010100:Oxygen; D002118:Calcium