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
N1 - Funding Information:
Acknowledgments This work was funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through grant DESC0010575 to KER. We thank Andrei Astashkin (Univ. of Arizona) for assistance with the EPR measurements and Patricia Baker for assistance with growing heliobacteria.
PY - 2014/6
Y1 - 2014/6
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
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U2 - 10.1007/s11120-014-9982-y
DO - 10.1007/s11120-014-9982-y
M3 - Article
C2 - 24557489
AN - SCOPUS:84901836472
SN - 0166-8595
VL - 120
SP - 291
EP - 299
JO - Photosynthesis research
JF - Photosynthesis research
IS - 3
ER -