M. Sugiura et al., Properties of Chlamydomonas photosystem II core complex with a His-tag at the C-terminus of the D2 protein, PLANT CEL P, 40(3), 1999, pp. 311-318
A His-tagged PSII core complex was purified from recombinant Chlamydomonas
reinhardtii D2-H thylakoids by single-step Ni2+-affinity column chromatogra
phy and its properties were partially characterized in terms of their PSII
functions and chemical compositions. The PSII core complex that has a His-t
ag extension at the C-terminus of the D2 protein evolved oxygen at a high r
ate of 2,400 mu mol (mg Chl)(-1) h(-1) at the optimum pH of 6.5 with ferric
yanide and 2,6-dichlorobenzoquinone as electron accepters in the presence o
f Ca2+ as an essential cofactor, and approximately 90% of the activity was
blocked by 10 mu M DCMU. The core complex exhibited the thermoluminescence
Q-band but not the B-band regardless of the presence or absence of DCMU, al
though both bands were observed in the His-tagged thylakoids. The core comp
lex was free from PSI and contained one Y-D, Tyr 160 of the D2 protein, fou
r Mn atoms, two cytochrome b-559, about 46 Chl a molecules, and probably on
e QA, the primary acceptor quinone of PSII. It was inferred from these resu
lts that His-tagging at the C-terminus of the D2 protein does not affect th
e functional and structural integrity of the PSII core complex, and that th
e 'His-tag strategy' is highly useful for biochemical, physicochemical, and
structural studies of Chlamydomonas PSII.