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 fromthe green sulfur bacterium Chlorobium tepidum by solubilizationof membranes with Triton X-100, followed by sucrosedensity gradientcentrifugation, DEAE Bio-Gel A chromatography, and hydroxyapatitechromatography. The purified RC complex contained about 50–70bacteriochlorophyll molecules (BChl) per P840, as assayed byphotooxidafion. It showed a near-infrared BChl a absorptionpeak at 814 nm and shoulders at about 800 and 835 nm at roomtemperature. SDS-PAGE analysis revealed 6 polypeptides withapparent molecular masses of 100, 65, 41, 32, 23, and 18 kDa.The RC complex binds functional P840 and Cyt c₅₅₁, which werephotooxidized by continuous illumination at room temperature.Upon flash excitation, the bound Cyt c₅₅₁ was oxidized, andrereduced in the dark with a half-time of 16 and 400 ms in thepresence and absence of 0.1 mM 2,6-dichlorophenol indophenol,respectively, at room temperature. At 551 nm, the amount ofthe Cyt c photooxidized by continuous illumination was 60% ofthe amount determined by chemical oxidation-reduction. The functionalCyt c₅₅₁/P840 ratio was calculated to be 1.2–1.7. EPRspectroscopy at cryogenic temperatures revealed that the RCcomplex binds three photoreducible Fe-S centers designated tobe CF_A, CF_B and CF_X (C for Chlorobium). CF_A and CF_B were reducedin the dark with dithionite at pH 10. (Received May 26, 1993; Accepted October 4, 1993)