Properties of Chlamydomonas photosystem II core complex with a his-tag at the C-terminus of the D2 protein
1999
Sugiura, M. (Institute of Physical and Chemical Research, Wako, Saitama (Japan)) | Minagawa, J. | Inoue, Y.
A His-tagged PSII core complex was purified from recombinant Chlamydomonas reinhardtii D2-H thylakoids by single-step Ni(2+)-affinity column chromatography and its properties were partially characterized in terms of their PSII functions and chemical compositions. The PSII core complex that has a His-tag extension at the C-terminus of the D2 protein evolved oxygen at a high rate of 2,400 mu-mol (mg Chl)(-1)(h-1) at the optimum pH of 6.5 with ferricyanide and 2,6-dichlorobenzoquinone as electron acceptors in the presence of Ca(2+) as an essential cofactor, and approximately 90% of the activity was blocked by 10 mu-M DCMU. The core complex exhibited the thermoluminescence Q-hand hut not the B-hand regardless of the presence or absence of DCMU, although both bands were observed in the His-tagged thylakoids. The core complex was free from PSI and contained one YD, Tyr 160 of the D2 protein, four Mn atoms, two cytochrome b-559, about 46 Chl a molecules, and probably one QA, the primary acceptor quinone. of PSII. It was inferred from these results that His-tagging at the C-terminus of the D2 protein does not affect the functional and structural integrity of the PSII core complex, and that the 'His-tag strategy' is highly useful for biochemical, physicochemical, and structural studies of Chlamydomonas PSII
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