Expression and characterization of cytochrome c 553 from Heliobacterium modesticaldum

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

Research output: Contribution to journalArticlepeer-review

7 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
Pages (from-to)291-299
Number of pages9
JournalPhotosynthesis Research
Volume120
Issue number3
DOIs
Publication statusPublished - Jun 2014

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Plant Science
  • Cell Biology

Fingerprint Dive into the research topics of 'Expression and characterization of cytochrome c <sub>553</sub> from Heliobacterium modesticaldum'. Together they form a unique fingerprint.

Cite this