PURIFICATION OF A PHOTOSYSTEM-II REACTION-CENTER FROM A THERMOPHILIC CYANOBACTERIUM USING IMMOBILIZED METAL AFFINITY-CHROMATOGRAPHY

Oxygen-evolving PS II particles from the thermophilic cyanobacterium S ynechococcus elongatus are partially purified by centrifugation on a s ucrose gradient and are bound to a Chelating Sepharose column loaded w ith Cu2+ ions. Bound particles are then transformed into PS II RC comp lexes by two washing steps. First, washing with a phosphate buffer (pH = 6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH = 6.2) containing 0.2 M LiClO4 and 0.05% of DM r emoves CP 47 and CP 43 and leaves bare PS II RC complexes on the colum n. These are then eluted with a phosphate buffer containing 1% of dode cylmaltoside (DM). The molar ratio of pigments in the eluate changes w ith the progress of elution but around the middle of the elution perio d a nearly stable ratio is maintained of Chi a : Pheo a : Car : Cyt b 559 equal to 2.9 : 1 : 0.9 : 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduce d pheophytin (Delta A of 430-440 nm) and by the flash induced formatio n of P680(+) (Delta A at 820 nm). The relatively slow relaxation kinet ics of the latter signal (t(1/2) approximate to 1 ms) may suggest that in a substantial fraction of the RCs Q(A) remains bound to the comple x.