title: Expression and characterization of cytochrome c553 from Heliobacterium modesticaldum.
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 × 10(4) 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.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, 85287-1604, USA.
authors.forename: Trevor S
authors.initials: TS
authors.lastname: Kashey
authors.forename: John B
authors.initials: JB
authors.lastname: Cowgill
authors.forename: Michael D
authors.initials: MD
authors.lastname: McConnell
authors.forename: Marco
authors.initials: M
authors.lastname: Flores
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Redding
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D045322:Photosynthetic Reaction Center Complex Proteins; D011994:Recombinant Proteins; C083951:cytochrome c553