Conformational change and human cytochrome c function: mutation of residue 41 modulates caspase activation and destabilizes Met-80 coordination

  • Tracy M. Josephs
  • Matthew D. Liptak
  • Gillian Hughes
  • Alexandra Lo
  • Rebecca M. Smith
  • Sigurd M. Wilbanks
  • Kara L. Bren
  • Elizabeth C. Ledgerwood
Original Paper

Abstract

Cytochrome c is a highly conserved protein, with 20 residues identical in all eukaryotic cytochromes c. Gly-41 is one of these invariant residues, and is the position of the only reported naturally occurring mutation in cytochrome c (human G41S). The basis, if any, for the conservation of Gly-41 is unknown. The mutation of Gly-41 to Ser enhances the apoptotic activity of cytochrome c without altering its role in mitochondrial electron transport. Here we have studied additional residue 41 variants and determined their effects on cytochrome c functions and conformation. A G41T mutation decreased the ability of cytochrome c to induce caspase activation and decreased the redox potential, whereas a G41A mutation had no impact on caspase induction but the redox potential increased. All residue 41 variants decreased the pK a of a structural transition of oxidized cytochrome c to the alkaline conformation, and this correlated with a destabilization of the interaction of Met-80 with the heme iron(III) at physiological pH. In reduced cytochrome c the G41T and G41S mutations had distinct effects on a network of hydrogen bonds involving Met-80, and in G41T the conformational mobility of two Ω-loops was altered. These results suggest the impact of residue 41 on the conformation of cytochrome c influences its ability to act in both of its physiological roles, electron transport and caspase activation.

Keywords

Cytochrome c Alkaline transition Apoptosis Redox potential NMR 

Abbreviations

Apaf-1

Apoptotic protease activating factor 1

CD

Circular dichroism

DSC

Differential scanning calorimetry

DTT

Dithiothreitol

NOE

Nuclear Overhauser enhancement

NOESY

Nuclear Overhauser enhancement spectroscopy

TOCSY

Total correlation spectroscopy

Notes

Acknowledgments

We gratefully acknowledge the Macromolecular Interactions Facility (The University of North Carolina at Chapel Hill) and the Centre for Protein Research (University of Otago) for resources. We thank Gary Pielak (Department of Chemistry, The University of North Carolina at Chapel Hill) and Ashutosh Tripathy (Macromolecular Interactions Facility, The University of North Carolina at Chapel Hill) for assistance with the DSC, Moira Hibbs (University of Otago) for technical assistance with protein expression and purification, and Rob Weeks (University of Otago) for assistance with cloning. This work was supported by the Health Research Council (New Zealand), the Marsden Fund (New Zealand), a University of Otago research grant, and the HS and JC Anderson Charitable Trusts (New Zealand) (E.C.L), an Elman Poole Travelling Scholarship (T.M.J.), and the National Institutes of Health (GM63170 to K.L.B and F32 GM089016 to M.D.L).

Supplementary material

775_2012_973_MOESM1_ESM.pdf (668 kb)
Supplementary material 1 (PDF 669 kb)

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Copyright information

© SBIC 2013

Authors and Affiliations

  • Tracy M. Josephs
    • 1
  • Matthew D. Liptak
    • 2
    • 3
  • Gillian Hughes
    • 1
  • Alexandra Lo
    • 1
  • Rebecca M. Smith
    • 2
  • Sigurd M. Wilbanks
    • 1
  • Kara L. Bren
    • 2
  • Elizabeth C. Ledgerwood
    • 1
  1. 1.Department of BiochemistryUniversity of OtagoDunedinNew Zealand
  2. 2.Department of ChemistryUniversity of RochesterRochesterUSA
  3. 3.Department of ChemistryUniversity of VermontBurlingtonUSA

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