, Volume 23, Issue 2, pp 239–248 | Cite as

Stability of cytochromes c′ from psychrophilic and piezophilic Shewanella species: implications for complex multiple adaptation to low temperature and high hydrostatic pressure

  • Asako Suka
  • Hiroya Oki
  • Yuki Kato
  • Kazuki Kawahara
  • Tadayasu Ohkubo
  • Takahiro Maruno
  • Yuji Kobayashi
  • Sotaro Fujii
  • Satoshi Wakai
  • Lisa Lisdiana
  • Yoshihiro SambongiEmail author
Original Paper


The stability of dimeric cytochrome c′ from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the heme and at the subunit–subunit interface, both of which are molecular interior regions. Here, we showed that interactions in the equivalent interior regions of homologous cytochromes c′ from two psychrophiles, Shewanella benthica and Shewanella violacea (SBCP and SVCP, respectively) were similarly weakened as compared with those of the counterparts of psychrophilic Shewanella livingstonensis and mesophilic Shewanella amazonensis (SLCP and SACP, respectively), and consistently the stability of SVCP, SLCP, and SACP increased in that order. Therefore, the stability of cytochromes c′ from the psychrophile, mesophile, and thermophile is systematically regulated in their molecular interior regions. Unexpectedly, however, the stability of SBCP was significantly higher than that of SVCP, and the former had additional molecular surface interactions. Collectively, SBCP had weakened interior interactions like SVCP did, but the former was stabilized at the molecular surface as compared with the latter, implying complex multiple adaptation of the proteins because the psychrophilic sources of SBCP and SVCP are also piezophilic, thriving in deep-sea extreme environments of low temperature and high hydrostatic pressure.


Cytochrome c′ Protein stability Piezophile Psychrophile Shewanella 



Circular dichroism


Shewanella amazonensis cytochrome c


Shewanella benthica cytochrome c


Shewanella livingstonensis cytochrome c


Shewanella violacea cytochrome c



We would like to thank Daisuke Yamane-Koshizawa for his technical support and the discussion.


This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (nos. 26240045 and 16K07692 to Y.S.), a Grant from the Japan Society for the Promotion of Science (no. 25–1446 to S.F.), a Grant-in-Aid for Young Human Resource Support from the Ministry of Education, Culture, Sports, Science and Technology in Japan (no. 1617PD0536 to S.F.), and a Grant-in-Aid for Fundamental Research from the Graduate School of Biosphere Science, Hiroshima University to S. F. L.L. is very grateful to the Indonesia Endowment Fund for Education, Ministry of Finance, Republic of Indonesia for the scholarship.


  1. Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung L, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NM, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zward PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr Sect D Biol Crystallogr 66:213–221CrossRefGoogle Scholar
  2. Akanuma S, Nakajima Y, Yokobori S, Kimura M, Nemoto N, Mase T, Miyazono K, Tanokura M, Yamagishi A (2013) Experimental evidence for the thermophilicity of ancestral life. Proc Natl Acad Sci USA 110:11067–11072CrossRefGoogle Scholar
  3. Ambler RP, Kamen MD, Bartsch RG, Meyer TE (1991) Amino acid sequences of Euglena viridis ferredoxin and cytochromes c. Biochem J 276:47–52CrossRefGoogle Scholar
  4. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: modeling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–W258CrossRefGoogle Scholar
  5. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66:12–21CrossRefGoogle Scholar
  6. Chou PY, Fasman GD (1978) Empirical predictions of protein conformation. Annu Rev Biochem 47:251–276CrossRefGoogle Scholar
  7. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132CrossRefGoogle Scholar
  8. Fujii S, Masanari M, Inoue H, Yamanaka M, Wakai S, Nishihara H, Sambongi Y (2013) High thermal stability and unique trimer formation of cytochrome c′ from thermophilic Hydrogenophilus thermoluteolus. Biosci Biotechnol Biochem 77:1677–1681CrossRefGoogle Scholar
  9. Fujii S, Oki H, Kawahara K, Yamane D, Yamanaka M, Maruno T, Kobayashi Y, Masanari M, Wakai S, Nishihara H, Ohkubo T, Sambongi Y (2017) Structural and functional insights into thermally stable cytochrome c′ from a thermophile. Protein Sci 26:737–748CrossRefGoogle Scholar
  10. Fujii S, Masanari-Fujii M, Kobayashi S, Kato C, Nishiyama M, Harada Y, Wakai S, Sambongi Y (2018) Commonly stabilized cytochromes c from deep-sea Shewanella and Pseudomonas. Biosci Biotechnol Biochem 82:792–799CrossRefGoogle Scholar
  11. Goihberg E, Dym O, Tel-Or S, Levin I, Peretz M, Burstein Y (2007) A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase. Proteins 66:196–204CrossRefGoogle Scholar
  12. Goto E, Kodama T, Minoda Y (1977) Isolation and culture conditions of thermophilic hydrogen bacteria. Agric Biol Chem 41:685–690Google Scholar
  13. Gromiha MM, Oobatake M, Sarai A (1999) Important amino acid properties for enhanced thermostability from mesophilic to thermophilic proteins. Biophys Chem 82:51–67CrossRefGoogle Scholar
  14. Hamajima Y, Nagae T, Watanabe N, Ohmae E, Kato-Yamada Y, Kato C (2016) Pressure adaptation of 3-isopropylmalate dehydrogenase from an extremely piezophilic bacterium is attributed to a single amino acid substitution. Extremophiles 20:177–186CrossRefGoogle Scholar
  15. Holm HW, Vennes JW (1970) Occurrence of purple sulfur bacteria in a sewage treatment lagoon. Appl Microbiol 19:988–996Google Scholar
  16. Inoue H, Wakai S, Nishihara H, Sambongi Y (2011) Heterologous synthesis of cytochrome c′ by Escherichia coli is not dependent on the System I cytochrome c biogenesis machinery. FEBS J 278:2341–2348CrossRefGoogle Scholar
  17. Kato C, Sato T, Horikoshi K (1995) Isolation and properties of barophilic and barotolerant bacteria from deep-sea mud samples barotolerant bacteria from deep-sea mud samples. Biodivers Conserv 4:1–9CrossRefGoogle Scholar
  18. Kato Y, Fujii S, Kuribayashi T, Masanari M, Sambongi Y (2015) Thermal stability of cytochrome c′ from mesophilic Shewanella amazonensis. Biosci Biotechnol Biochem 79:1125–1129CrossRefGoogle Scholar
  19. Kulakova L, Galkin A, Kurihara T, Yoshimura T, Esaki N (1999) Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain Ac10: gene cloning and enzyme purification and characterization. Appl Environ Microbiol 65:611–617Google Scholar
  20. Kuribayashi T, Fujii S, Masanari M, Yamanaka M, Wakai S, Sambongi Y (2017) Difference in NaCl tolerance of membrane-bound 5’-nucleotidases purified from deep-sea and brackish water Shewanella species. Extremophiles 21:357–368CrossRefGoogle Scholar
  21. Lauro FM, Chastain RA, Ferriera S, Johnson J, Yayanos AA, Bartlett DH (2013) Draft genome sequence of the deep-sea bacterium Shewanella benthica strain KT99. Genome Announc 1:e00210–e00213Google Scholar
  22. Lee CF, Makhatadze GI, Wong KB (2005) Effects of charge-to-alanine substitutions on the stability of ribosomal protein L30e from Thermococcus celer. Biochemistry 44:16817–16825CrossRefGoogle Scholar
  23. Manole A, Kekilli D, Svistunenko DA, Wilson MT, Dobbin PS, Hough MA (2015) Conformational control of the binding of diatomic gases to cytochrome c′. J Biol Inorg Chem 20:675–686CrossRefGoogle Scholar
  24. Masanari M, Wakai S, Ishida M, Kato C, Sambongi Y (2014) Correlation between the optimal growth pressures of four Shewanella species and the stabilities of their cytochromes c 5. Extremophiles 18:617–627CrossRefGoogle Scholar
  25. Matthews BM (1968) Solvent content of protein crystals. J Mol Biol 33:491–497CrossRefGoogle Scholar
  26. Matthews BW, Nicholson H, Becktel WJ (1987) Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci USA 84:6663–6667CrossRefGoogle Scholar
  27. McCoy AJ, Grosse-Kunstleve RW, Adams PD, WinnMD Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674CrossRefGoogle Scholar
  28. Moore GR (1991) Bacterial 4-alpha-helical bundle cytochromes. Biochim Biophys Acta 1058:38–41CrossRefGoogle Scholar
  29. Perl D, Mueller U, Heinemann U, Schmid FX (2000) Two exposed amino acid residues confer thermostability on a cold shock protein. Nat Struct Biol 7:380–383CrossRefGoogle Scholar
  30. Sambongi Y, Ferguson SJ (1994) Synthesis of holo Paracoccus denitrificans cytochrome c 550 requires targeting to the periplasm whereas that of holo Hydrogenobacter thermophilus cytochrome c 552 does not. Implications for c-type cytochrome biogenesis. FEBS Lett 340:65–70CrossRefGoogle Scholar
  31. Sambongi Y, Stoll R, Ferguson SJ (1996) Alteration of haem-attachment and signal-cleavage sites for Paracoccus denitrificans cytochrome c 550 probes pathway of c-type cytochrome biogenesis in Escherichia coli. Mol Microbiol 19:1193–1204CrossRefGoogle Scholar
  32. Sambongi Y, Uchiyama S, Kobayashi Y, Igarashi Y, Hasegawa J (2002) Cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability. Eur J Biochem 269:3355–3361CrossRefGoogle Scholar
  33. Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J 78:1606–1619CrossRefGoogle Scholar
  34. Scopes RK (1974) Measurement of protein by spectrophotometry at 205 nm. Anal Biochem 59:277–282CrossRefGoogle Scholar
  35. Sriprapundh D, Vieille C, Zeikus JG (2000) Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis. Protein Eng 13:259–265CrossRefGoogle Scholar
  36. Takenaka S, Wakai S, Tamegai H, Uchiyama S, Sambongi Y (2010) Comparative analysis of highly homologous Shewanella cytochromes c 5 for stability and function. Biosci Biotechnol Biochem 74:1079–1083CrossRefGoogle Scholar
  37. Uchiyama S, Ohshima A, Nakamura S, Hasegawa J, Terui N, Takayama SJ, Yamamoto Y, Sambongi Y, Kobayashi Y (2004) Complete thermal-unfolding profiles of oxidized and reduced cytochromes c. J Am Chem Soc 126:14684–14685CrossRefGoogle Scholar
  38. Venkateswaran K, Dollhopf ME, Aller R, Stackebrandt E, Nealson KH (1998) Shewanella amazonensis sp. nov., a novel metal-reducing facultative anaerobe from Amazonian shelf muds. Int J Syst Bacteriol 48:965–972CrossRefGoogle Scholar
  39. Yamane-Koshizawa D, Fujii S, Maruno T, Kobayashi Y, Yamanaka M, Wakai S, Sambongi Y (2018) Stabilization of mesophilic Allochromatium vinosum cytochrome c′ through specific mutations modeled by a thermophilic homologue. Biosci Biotechnol Biochem 82:304–311CrossRefGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  • Asako Suka
    • 1
  • Hiroya Oki
    • 2
  • Yuki Kato
    • 1
  • Kazuki Kawahara
    • 2
  • Tadayasu Ohkubo
    • 2
  • Takahiro Maruno
    • 3
  • Yuji Kobayashi
    • 3
  • Sotaro Fujii
    • 1
  • Satoshi Wakai
    • 4
  • Lisa Lisdiana
    • 1
    • 5
  • Yoshihiro Sambongi
    • 1
    Email author
  1. 1.Graduate School of Biosphere ScienceHiroshima UniversityHiroshimaJapan
  2. 2.Graduate School of Pharmaceutical SciencesOsaka UniversitySuitaJapan
  3. 3.Graduate School of EngineeringOsaka UniversitySuitaJapan
  4. 4.Graduate School of Science, Technology, and InnovationKobe UniversityKobeJapan
  5. 5.Department of BiologyUniversitas Negeri SurabayaSurabayaIndonesia

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