STABLE PHOTOBLEACHING OF P840 IN CHLOROBIUM REACTION-CENTER PREPARATIONS - PRESENCE OF THE 42-KDA BACTERIOCHLOROPHYLL ALPHA-PROTEIN AND A 17-KDA POLYPEPTIDE

Simple procedures for the anaerobic preparation of photoactive and sta ble P840 reaction centers from Chlorobium tepidum and Chlorobium limic ola in good yield are presented and quantitated. The subunit compositi on was tested by cosedimentation in sucrose density gradients. For C. limicola, it minimally comprises four subunits: the P840 reaction cent er protein PscA, the BChla antenna protein FMO, the FeS protein PscB w ith centers A and B, and a positively charged 17-kDa protein denoted P scD, The preparation from Chlorobium tepidum additionally contained Ps cC, a cytochrome c-551. The BChla absorption peak of the purified comp lexes was at 810 nm, with a shoulder at 835 nm. The ratio of the shoul der to the peak was 0.25, which corresponds to 1 reaction center per 7 0 BChla molecules if a uniform extinction coefficient of BChla is assu med. However, bleaching at 610 nm in continuous light corresponded up to 1 photoactive reaction center per 50 BChla molecules. Therefore, ei ther the extinction coefficient of BChla in the reaction center is ove restimated or the one for photobleaching is underestimated. In any cas e, the major portion of the reaction center was photoactive in the pre parations. A P840 reaction center subcomplex, lacking PscD and deficie nt in FMO and PscB, but retaining the cytochrome c subunit, was obtain ed as a side product. It was photoinactive and had an absorption peak at 814 nm and a 835/814 absorbance ratio of 0.42. FMO and PscB show th e tendency to form a complementary subcomplex. FMO and PscD are appare ntly required to stabilize the photoactive reaction center, while the cytochrome c subunit is not.