Expression and characterization of cytochrome c 553 from Heliobacterium modesticaldum

Trevor S. Kashey, John B. Cowgill, Michael D. McConnell, Marco Flores, Kevin Redding

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    Cytochrome c553 of Heliobacterium modesticaldum is the donor to P800 +, the primary electron donor of the heliobacterial reaction center (HbRC). It is a membrane-anchored 14-kDa cytochrome that accomplishes electron transfer from the cytochrome bc complex to the HbRC. The petJ gene encoding cyt c 553 was cloned and expressed in Escherichia coli with a hexahistidine tag replacing the lipid attachment site to create a soluble donor that could be made in a preparative scale. The recombinant cytochrome had spectral characteristics typical of a c-type cytochrome, including an asymmetric α-band, and a slightly red-shifted Soret band when reduced. The EPR spectrum of the oxidized protein was characteristic of a low-spin cytochrome. The midpoint potential of the recombinant cytochrome was +217 ± 10 mV. The interaction between soluble recombinant cytochrome c 553 and the HbRC was also studied. Re-reduction of photooxidized P800 + was accelerated by addition of reduced cytochrome c 553. The kinetics were characteristic of a bimolecular reaction with a second order rate of 1.53 × 104 M-1 s -1 at room temperature. The rate manifested a steep temperature dependence, with a calculated activation energy of 91 kJ mol-1, similar to that of the native protein in Heliobacillus gestii cells. These data demonstrate that the recombinant soluble cytochrome is comparable to the native protein, and likely lacks a discrete electrostatic binding site on the HbRC.

    Original languageEnglish (US)
    Pages (from-to)291-299
    Number of pages9
    JournalPhotosynthesis Research
    Volume120
    Issue number3
    DOIs
    StatePublished - 2014

    Fingerprint

    Heliobacterium
    cytochrome c
    Cytochromes
    cytochromes
    Cytochromes c
    His-His-His-His-His-His
    Heliobacillus
    Cytochrome c Group
    Electrons
    Proteins
    Temperature
    Gene encoding
    Static Electricity
    Escherichia coli
    Paramagnetic resonance
    proteins
    Electrostatics
    Activation energy
    activation energy
    Binding Sites

    Keywords

    • Cytochrome c
    • Heliobacterium modesticalcum
    • Photosynthetic reaction center
    • Transient spectroscopy

    ASJC Scopus subject areas

    • Plant Science
    • Cell Biology
    • Biochemistry
    • Medicine(all)

    Cite this

    Expression and characterization of cytochrome c 553 from Heliobacterium modesticaldum. / Kashey, Trevor S.; Cowgill, John B.; McConnell, Michael D.; Flores, Marco; Redding, Kevin.

    In: Photosynthesis Research, Vol. 120, No. 3, 2014, p. 291-299.

    Research output: Contribution to journalArticle

    Kashey, Trevor S. ; Cowgill, John B. ; McConnell, Michael D. ; Flores, Marco ; Redding, Kevin. / Expression and characterization of cytochrome c 553 from Heliobacterium modesticaldum. In: Photosynthesis Research. 2014 ; Vol. 120, No. 3. pp. 291-299.
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