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

Noriaki Kusumoto, Kazuhito Inoue, Hidekazu Nasu, Hidehiro Sakurai

    Research output: Contribution to journalArticle

    41 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)17-25
    Number of pages9
    JournalPlant and Cell Physiology
    Volume35
    Issue number1
    Publication statusPublished - 1994

    Fingerprint

    Chlorobium
    Chlorobium tepidum
    Chlorobi
    Reaction Center
    cytochrome c
    Cytochromes c
    Bacteria
    cytochrome
    Preparation
    ambient temperature
    Sulfur
    sulfur
    Bacteriochlorophylls
    Proteins
    bacterium
    Temperature
    lighting
    chromatography
    Chromatography
    Lighting

    Keywords

    • Chlorobium tepidum
    • Cytochrome c
    • Green sulfur bacterium
    • Iron-sulfur center
    • P840
    • Reaction center

    ASJC Scopus subject areas

    • Statistics, Probability and Uncertainty
    • Applied Mathematics
    • Ecology
    • Cell Biology
    • Physiology
    • Plant Science

    Cite this

    Preparation of a photoactive reaction center complex containing photo-reducible fe-s centers and photooxidizable cytochrome c from the green sulfur bacterium chlorobium tepidum. / Kusumoto, Noriaki; Inoue, Kazuhito; Nasu, Hidekazu; Sakurai, Hidehiro.

    In: Plant and Cell Physiology, Vol. 35, No. 1, 1994, p. 17-25.

    Research output: Contribution to journalArticle

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