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Roles of a short connecting disulfide bond in the stability and function of psychrophilic Shewanella violacea cytochrome c 5*

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Abstract

Cys-59 and Cys-62, forming a disulfide bond in the four-residue loop of Shewanella violacea cytochrome c 5 (SV cytc 5), contribute to protein stability but not to redox function. These Cys residues were substituted with Ala in SV cytc 5, and the structural and functional properties of the resulting C59A/C62A variant were determined and compared with those of the wild-type. The variant had similar features to those of the wild-type in absorption, circular dichroic, and paramagnetic 1H NMR spectra. In addition, the redox potentials of the wild-type and variant were essentially the same, indicating that removal of the disulfide bond from SV cytc 5 does not affect the redox function generated in the vicinity of heme. However, calorimetric analysis of the wild-type and variant showed that the mutations caused a drastic decrease in the protein stability through enthalpy, but not entropy. Four residues are encompassed by the SV cytc 5 disulfide bond, which is the shortest one that has been proved to affect protein stability. The protein stability of SV cytc 5 can be controlled without changing the redox function, providing a new strategy for regulating the stability and function of cytochrome c.

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Abbreviations

SV cytc 5 :

Shewanella violacea mono-heme cytochrome c5

CD:

Circular dichroism

CV:

Cyclic voltammetry

DSC:

Differential scanning calorimetry

PCR:

Polymerase chain reaction

SDS:

Sodium dodecyl sulfate

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Acknowledgments

This work was financially supported in part by grants from the Japanese Ministry of Education, Science and Culture (Grants-in-Aid for Scientific Research on Priority Areas) to Y. S. and Y.Y. (17350081), the Yazaki Memorial Foundation for Science and Technology, and the NOVARTIS Foundation (Japan) for the Promotion of Science to Y.Y.

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Authors and Affiliations

  1. Graduate School of Biosphere Science, Hiroshima University, CREST of Japan Science and Technology Corporation, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8528, Japan

    Keiko Ogawa, Takafumi Sonoyama, Taku Takeda, Shin-ichi Ichiki & Yoshihiro Sambongi

  2. Graduate School of Pharmaceutical Sciences, Osaka University, Suita, 565-0871, Japan

    Shota Nakamura & Yuji Kobayashi

  3. Graduate School of Engineering, Osaka University, Suita, 565-0871, Japan

    Susumu Uchiyama

  4. Department of Chemistry and Environmental Technology, School of Engineering, Kinki University, Umenobe Takaya 1, Higashi-Hiroshima, Hiroshima, 739-2116, Japan

    Kaoru Nakasone

  5. Department of Chemistry, University of Tsukuba, Tsukuba, 305-8571, Japan

    Shin-ichi J. Takayama, Hajime Mita & Yasuhiko Yamamoto

Authors
  1. Keiko Ogawa
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  2. Takafumi Sonoyama
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  3. Taku Takeda
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  4. Shin-ichi Ichiki
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  5. Shota Nakamura
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  6. Yuji Kobayashi
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  7. Susumu Uchiyama
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  8. Kaoru Nakasone
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  9. Shin-ichi J. Takayama
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  10. Hajime Mita
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  11. Yasuhiko Yamamoto
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  12. Yoshihiro Sambongi
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Corresponding author

Correspondence to Yoshihiro Sambongi.

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Communicated by K. Horikoshi.

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Ogawa, K., Sonoyama, T., Takeda, T. et al. Roles of a short connecting disulfide bond in the stability and function of psychrophilic Shewanella violacea cytochrome c 5*. Extremophiles 11, 797–807 (2007). https://doi.org/10.1007/s00792-007-0099-5

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  • DOI: https://doi.org/10.1007/s00792-007-0099-5

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