Abstract
Cytochrome c z is found in green sulfur photosynthetic bacteria, and is considered to be the only electron donor to the special pair P840 of the reaction center. It consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. Large scale expression of the C-terminal functional domain of the cytochrome c z (C-cyt c z) from the thermophilic bacterium Chlorobium tepidum has been achieved using the Escherichia coli expression system. The C-cyt c z expressed has been highly purified, and is stable at room temperature over 10 days of incubation for both reduced and oxidized forms. Spectroscopic measurements indicate that the heme iron in C-cyt c z is in a low-spin state and this does not change with the redox state. 1H-NMR spectra of the oxidized C-cyt c z exhibited unusually large paramagnetic chemical shifts for the heme methyl protons in comparison with those of other Class I ferric cytochromes c. Differences in the 1H-NMR linewidth were observed for some resonances, indicating different dynamic environments for these protons. Crystals of the oxidized C-cyt c z were obtained using ammonium sulfate as a precipitant. The crystals diffracted X-rays to a maximum resolution of 1.2 Å, and the diffraction data were collected to 1.3 Å resolution.
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Abbreviations
- C-cyt c z :
-
C-terminal domain of the cytochrome c z
- Chl :
-
Chlorobium
- CD:
-
Circular dichroism
- MALDI–TOF:
-
Matrix-assisted laser desorption/ionization time-of-flight
- MCD:
-
Magnetic circular dichroism
- PCR:
-
Polymerase chain reaction
- PEG:
-
Polyethylene glycol
- RC:
-
Reaction center
- RR:
-
Resonance Raman
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Acknowledgments
We thank Dr. L. Thöny-Meyer for providing us with pEC86 and Dr. T. Kohzuma for the MALDI–TOF/MS measurement. This study has been performed under the approval of the Photon Factory Program Advisory Committee (Proposal No. 2007G585), and was supported by grants-in-aid for Scientific Research on Priority Areas “Structures of Biological Macromolecular Assemblies” and by The Kurata Memorial Hitachi Science and Technology Foundation.
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Higuchi, M., Hirano, Y., Kimura, Y. et al. Overexpression, characterization, and crystallization of the functional domain of cytochrome c z from Chlorobium tepidum . Photosynth Res 102, 77–84 (2009). https://doi.org/10.1007/s11120-009-9492-5
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DOI: https://doi.org/10.1007/s11120-009-9492-5