Preparation of a photoactive reaction center complex containing photo-reducible fe-s centers and photooxidizable cytochrome c from the green sulfur bacterium chlorobium tepidum

A photoactive reaction center (RC) complex was isolated from the green sulfur bacterium Chlorobium tepidum by solubilization of membranes with Triton X-100, followed by sucrosedensity gradient centrifugation, DEAE Bio-Gel A chromatography, and hydroxyapatite chromatography. The purified RC complex contained about 50-70 bacteriochlorophyll molecules (BChl) per P840, as assayed by photooxidafion. It showed a near-infrared BChl a absorption peak at 814 nm and shoulders at about 800 and 835 nm at room temperature. SDS-PAGE analysis revealed 6 polypeptides with apparent molecular masses of 100, 65, 41, 32, 23, and 18 kDa. The RC complex binds functional P840 and Cyt c₅₅₁, which were photooxidized by continuous illumination at room temperature. Upon flash excitation, the bound Cyt c₅₅₁ was oxidized, and rereduced in the dark with a half-time of 16 and 400 ms in the presence and absence of 0.1 mM 2,6-dichlorophenol indophenol, respectively, at room temperature. At 551 nm, the amount of the Cyt c photooxidized by continuous illumination was 60% of the amount determined by chemical oxidation-reduction. The functional Cyt c₅₅₁/P840 ratio was calculated to be 1.2-1.7. EPR spectroscopy at cryogenic temperatures revealed that the RC complex binds three photoreducible Fe-S centers designated to be CF_A, CF_B and CF_X (C for Chlorobium). CF_A and CF_B were reduced in the dark with dithionite at pH 10.