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

6 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.
@article{11a18c30b1124c7f9c137fe386c025ea,
title = "Expression and characterization of cytochrome c 553 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 × 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.",
keywords = "Cytochrome c, Heliobacterium modesticalcum, Photosynthetic reaction center, Transient spectroscopy",
author = "Kashey, {Trevor S.} and Cowgill, {John B.} and McConnell, {Michael D.} and Marco Flores and Kevin Redding",
year = "2014",
doi = "10.1007/s11120-014-9982-y",
language = "English (US)",
volume = "120",
pages = "291--299",
journal = "Photosynthesis Research",
issn = "0166-8595",
publisher = "Springer Netherlands",
number = "3",

}

TY - JOUR

T1 - Expression and characterization of cytochrome c 553 from Heliobacterium modesticaldum

AU - Kashey, Trevor S.

AU - Cowgill, John B.

AU - McConnell, Michael D.

AU - Flores, Marco

AU - Redding, Kevin

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - Cytochrome c

KW - Heliobacterium modesticalcum

KW - Photosynthetic reaction center

KW - Transient spectroscopy

UR - http://www.scopus.com/inward/record.url?scp=84901836472&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84901836472&partnerID=8YFLogxK

U2 - 10.1007/s11120-014-9982-y

DO - 10.1007/s11120-014-9982-y

M3 - Article

C2 - 24557489

AN - SCOPUS:84901836472

VL - 120

SP - 291

EP - 299

JO - Photosynthesis Research

JF - Photosynthesis Research

SN - 0166-8595

IS - 3

ER -