Journal of Biomolecular NMR

, Volume 52, Issue 3, pp 245–256 | Cite as

Redox-dependent conformational changes in eukaryotic cytochromes revealed by paramagnetic NMR spectroscopy

  • Alexander N. Volkov
  • Sophie Vanwetswinkel
  • Karen Van de Water
  • Nico A. J. van Nuland


Cytochrome c (Cc) is a soluble electron carrier protein, transferring reducing equivalents between Cc reductase and Cc oxidase in eukaryotes. In this work, we assessed the structural differences between reduced and oxidized Cc in solution by paramagnetic NMR spectroscopy. First, we have obtained nearly-complete backbone NMR resonance assignments for iso-1-yeast Cc and horse Cc in both oxidation states. These were further used to derive pseudocontact shifts (PCSs) arising from the paramagnetic haem group. Then, an extensive dataset comprising over 450 measured PCSs and high-resolution X-ray and solution NMR structures of both proteins were used to define the anisotropic magnetic susceptibility tensor, Δχ. For most nuclei, the PCSs back-calculated from the Δχ tensor are in excellent agreement with the experimental PCS values. However, several contiguous stretches—clustered around G41, N52, and A81—exhibit large deviations both in yeast and horse Cc. This behaviour is indicative of redox-dependent structural changes, the extent of which is likely conserved in the protein family. We propose that the observed discrepancies arise from the changes in protein dynamics and discuss possible functional implications.


Cytochrome c Paramagnetic NMR Protein dynamics Pseudocontact shifts 



We thank Dr. Jonathan Worrall and Prof. Marcellus Ubbink for the kind gifts of the Cc expression vectors, Dr. Christophe Schmitz for useful tips on the installation and use of Numbat software, and Dr. Lieven Buts for the help with the data analysis. ANV is an FWO Post-Doctoral Researcher.

Supplementary material

10858_2012_9607_MOESM1_ESM.docx (1.1 mb)
Supplementary material 1 (DOCX 1126 kb)


  1. Baistrocchi P, Banci L, Bertini I, Turano P, Bren KL, Gray HB (1996) Three-dimensional solution structure of Saccharomyces cerevisiae reduced iso-1-cytochrome c. Biochemistry 35:13788–13796CrossRefGoogle Scholar
  2. Banci L, Assfalg M (2001) Mitochondrial cytochrome c. In: Messerschmidt A, Huber R, Poulos TL, Wieghardt K (eds) Handbook of metalloproteins. Wiley, Chichester, pp 33–43Google Scholar
  3. Banci L, Bertini I, Bren KL, Gray HB, Sompornpisut P, Turano P (1997a) Solution structure of oxidized Saccharomyces cerevisiae iso-1-cytochrome c. Biochemistry 36:8992–9001CrossRefGoogle Scholar
  4. Banci L, Bertini I, Gray HB, Luchinat C, Reddig T, Rosato A, Turano P (1997b) Solution structure of oxidized horse heart cytochrome c. Biochemistry 36:9867–9877CrossRefGoogle Scholar
  5. Banci L, Bertini I, Huber JG, Spyroulias GA, Turano P (1999a) Solution structure of reduced horse heart cytochrome c. J Biol Inorg Chem 4:21–31CrossRefGoogle Scholar
  6. Banci L, Bertini I, Rosato A, Varani G (1999b) Mitochondrial cytochromes c: a comparative analysis. J Biol Inorg Chem 4:824–837CrossRefGoogle Scholar
  7. Banci L, Bertini I, Luchinat C, Turano P (2000) Solution structures of hemoproteins. In: Kadish KM, Smith KM, Guilard R (eds) The porphyrin handbook. Academic Press, San Diego, CA, pp 323–350Google Scholar
  8. Baxter SM, Fetrow JS (1999) Hydrogen exchange behavior of [U-15N]-labeled oxidised and reduced iso-1-cytochrome c. Biochemistry 38:4493–4503CrossRefGoogle Scholar
  9. Berghuis AM, Brayer GD (1992) Oxidation state-dependent conformational changes in cytochrome c. J Mol Biol 223:959–976CrossRefGoogle Scholar
  10. Bertini I, Luchinat C, Parigi G (2002) Magnetic susceptibility in paramagnetic NMR. Progress in Nuclear Magnetic Resonance Spectroscopy 40:249–273CrossRefGoogle Scholar
  11. Boyd J, Dobson CM, Morar AS, Williams RJP, Pielak GJ (1999) 1H and 15N hyperfine shifts of cytochrome c. J Am Chem Soc 121:9247–9248CrossRefGoogle Scholar
  12. Bushnell GW, Louie GV, Brayer GD (1990) High-resolution three-dimensional structure of horse heart cytochrome c. J Mol Biol 214:585–595CrossRefGoogle Scholar
  13. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  14. DeLano WL (2002) The PyMOL molecular graphics system. DeLano Scientific, Palo AltoGoogle Scholar
  15. Feng YQ, Roder H, Englander SW (1990) Redox-dependent structure change and hyperfine nuclear magnetic resonance shifts in cytochrome c. Biochemistry 29:3494–3504CrossRefGoogle Scholar
  16. Fetrow JS, Baxter SM (1999) Assignment of 15N chemical shifts and 15N relaxation measurements for oxidized and reduced iso-1-cytochrome c. Biochemistry 38:4480–4492CrossRefGoogle Scholar
  17. Gao Y, Boyd J, Williams RJP, Pielak GJ (1990) Assignment of proton resonances, identification of secondary structural elements, and analysis of backbone chemical shifts for the C102T variant of yeast iso-1-cytochrome c and horse cytochrome c. Biochemistry 29:6994–7003CrossRefGoogle Scholar
  18. Gao Y, Boyd J, Pielak GJ, Williams RJP (1991) Comparison of reduced and oxidized yeast iso-1-cytochrome c using proton paramagnetic shifts. Biochemistry 30:1928–1934CrossRefGoogle Scholar
  19. Han B, Liu Y, Ginzinger S, Wishart D (2011) SHIFTX2: significantly improved protein chemical shift prediction. J Biomol NMR 50:43–57CrossRefGoogle Scholar
  20. Iwahara J, Schwieters CD, Clore GM (2004) Ensemble approach for NMR structure refinement against 1H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule. J Am Chem Soc 126:5879–5896CrossRefGoogle Scholar
  21. John M, Park AY, Pintacuda G, Dixon NE, Otting G (2005) Weak alignment of paramagnetic proteins warrants correction for residual CSA effects in measurements of pseudocontact shifts. J Am Chem Soc 127:17190–17191CrossRefGoogle Scholar
  22. Levitt M, Perutz MF (1988) Aromatic rings act as hydrogen bond acceptors. J Mol Biol 201:751–754CrossRefGoogle Scholar
  23. Liu W, Rumbley J, Englander SW, Wand AJ (2003) Backbone and side-chain heteronuclear resonance assignments and hyperfine NMR shifts in horse cytochrome c. Prot Sci 12:2104–2108CrossRefGoogle Scholar
  24. Louie GV, Brayer GD (1990) High-resolution refinement of yeast iso-1-cytochrome c and comparisons with other eukaryotic cytochromes c. J Mol Biol 214:527–555CrossRefGoogle Scholar
  25. Moench SJ, Shi TM, Satterlee JD (1991) Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c. Eur J Biochem 197:631–641CrossRefGoogle Scholar
  26. Morar AS, Kakouras D, Young GB, Boyd J, Pielak GJ (1999) Expression of 15N-labeled eukaryotic cytochrome c in Escherichia coli. J Biol Inorg Chem 4:220–222CrossRefGoogle Scholar
  27. Pollock WBR, Rosell FI, Twitchett MB, Dumont ME, Mauk AG (1998) Bacterial expression of a mitochondrial cytochrome c. Trimethylation of Lys 72 in yeast iso-1-cytochrome c and the alkaline conformational transition. Biochemistry 37:6124–6131CrossRefGoogle Scholar
  28. Qi PX, Beckman RA, Wand AJ (1996) Solution structure of horse heart ferricytochrome c and detection of redox-related structural changes by high-resolution 1H NMR. Biochemistry 35:12275–12286CrossRefGoogle Scholar
  29. Rule GS, Hitchens TK (2006) Fundamentals of protein NMR spectroscopy. Springer, DordrechtGoogle Scholar
  30. Rumbley JN, Hoang L, Englander SW (2002) Recombinant equine cytochrome c in Escherichia coli: high-level expression, characterization, and folding and assembly mutants. Biochemistry 41:13894–13901CrossRefGoogle Scholar
  31. Schmitz C, Stanton-Cook MJ, Su X-C, Otting G, Huber T (2008) Numbat: an interactive software tool for fitting Δχ-tensors to molecular coordinates using pseudocontact shifts. J Biomol NMR 41:179–189CrossRefGoogle Scholar
  32. Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM (2003) The Xplor-NIH NMR molecular structure determination package. J Magn Reson 160:66–74ADSCrossRefGoogle Scholar
  33. Sukits SF, Erman JE, Satterlee JD (1997) Proton NMR assignments and magnetic axes orientations for wild-type yeast iso-1-ferricytochrome c free in solution and bound to cytochrome c peroxidase. Biochemistry 36:5251–5259CrossRefGoogle Scholar
  34. Timkovich R, Cai M (1993) Investigation of the structure of oxidized Pseudomonas aeruginosa cytochrome c-551 by NMR: comparison of observed paramagnetic shifts and calculated pseudocontact shifts. Biochemistry 32:11516–11523CrossRefGoogle Scholar
  35. Turner DL, Williams RJP (1993) 1H and 13C NMR investigation of redox-state-dependent and temperature-dependent conformational changes in horse cytochrome c. Eur J Biochem 211:555–562CrossRefGoogle Scholar
  36. Ubbink M, Worrall JAR, Canters GW, Groenen EJJ, Huber M (2002) Paramagnetic resonance of biological metal centers. Ann Rev Biophys Biomol Struc 31:393–422CrossRefGoogle Scholar
  37. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59:687–696CrossRefGoogle Scholar
  38. Weiner MP, Costa GL, Schoettlin W, Cline J, Mathur E, Bauer JC (1994) Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene 151:119–123CrossRefGoogle Scholar
  39. Williams G, Clayden NJ, Moore GR, Williams RJP (1985) Comparison of the solution and crystal structures of mitochondrial cytochrome c—analysis of paramagnetic shifts in the nuclear magnetic resonance spectrum of ferricytochrome c. J Mol Biol 183:447–460CrossRefGoogle Scholar
  40. Worrall JAR, Kolczak U, Canters GW, Ubbink M (2001) Interaction of yeast iso-1-cytochrome c with cytochrome c peroxidase investigated by [15N, 1H] heteronuclear NMR spectroscopy. Biochemistry 40:7069–7076CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Alexander N. Volkov
    • 1
    • 2
  • Sophie Vanwetswinkel
    • 1
    • 2
  • Karen Van de Water
    • 1
    • 2
  • Nico A. J. van Nuland
    • 1
    • 2
  1. 1.Jean Jeener NMR Centre, Structural Biology BrusselsVrije Universiteit BrusselBrusselsBelgium
  2. 2.Department of Structural BiologyVIBBrusselsBelgium

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