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Noriaki Kusumoto, Kazuhito Inoue, Hidekazu Nasu, Hidehiro Sakurai, Preparation of a Photoactive Reaction Center Complex Containing Photo-reducible Fe-S Centers and Photooxidizable Cytochrome c from the Green Sulfur Bacterium Chlorobium tepidum, Plant and Cell Physiology, Volume 35, Issue 1, 1994, Pages 17–25, https://doi.org/10.1093/oxfordjournals.pcp.a078566
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Abstract
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 CFA, CFB and CFX (C for Chlorobium). CFA and CFB were reduced in the dark with dithionite at pH 10.
