Properties of Chlamydomonas Photosystem II Core Complex with a His-Tag at the C-Terminus of the D2 Protein

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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 μmol (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 μM DCMU. The core complex exhibited the thermoluminescence Q-band but not the B-band 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 Y_D, Tyr 160 of the D2 protein, four Mn atoms, two cytochrome b-559, about 46 Chl a molecules, and probably one Q_A, the primary acceptor quinone of PSII. It was inferred from these results that His-tagging at the C-terminus of that D2 protein does not affect the functional and structural integrity of the PSII core complex, and the the 'His-tag strategy' is highly useful for biochemical, physicochemical, and structural studies of Chlamydomonas PSII.
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