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.
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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 DE-SC0010575 to KER. We thank Andrei Astashkin (Univ. of Arizona) for assistance with the EPR measurements and Patricia Baker for assistance with growing heliobacteria.
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Kashey, T.S., Cowgill, J.B., McConnell, M.D. et al. Expression and characterization of cytochrome c 553 from Heliobacterium modesticaldum . Photosynth Res 120, 291–299 (2014). https://doi.org/10.1007/s11120-014-9982-y
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DOI: https://doi.org/10.1007/s11120-014-9982-y