Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only

The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axi...

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Veröffentlicht in:Biochemistry (Easton) Jg. 56; H. 13; S. 1887
Hauptverfasser: Milazzo, Lisa, Tognaccini, Lorenzo, Howes, Barry D, Sinibaldi, Federica, Piro, Maria C, Fittipaldi, Maria, Baratto, Maria C, Pogni, Rebecca, Santucci, Roberto, Smulevich, Giulietta
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Sprache:Englisch
Veröffentlicht: United States 04.04.2017
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ISSN:1520-4995, 1520-4995
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Abstract The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH ion.
AbstractList The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH- ion.The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH- ion.
The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH ion.
Author Tognaccini, Lorenzo
Baratto, Maria C
Fittipaldi, Maria
Pogni, Rebecca
Sinibaldi, Federica
Smulevich, Giulietta
Piro, Maria C
Santucci, Roberto
Milazzo, Lisa
Howes, Barry D
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  surname: Milazzo
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  givenname: Lorenzo
  surname: Tognaccini
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  organization: Dipartimento di Chimica "Ugo Schiff", Università di Firenze , Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy
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  givenname: Barry D
  surname: Howes
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  givenname: Federica
  surname: Sinibaldi
  fullname: Sinibaldi, Federica
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  organization: Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena , Via Aldo Moro 2, 53100 Siena, Italy
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  givenname: Rebecca
  surname: Pogni
  fullname: Pogni, Rebecca
  organization: Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena , Via Aldo Moro 2, 53100 Siena, Italy
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  surname: Santucci
  fullname: Santucci, Roberto
  organization: Dipartimento di Scienze Cliniche e Medicina Traslazionale, Università di Roma "Tor Vergata" , Via Montpellier 1, 00133 Rome, Italy
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  givenname: Giulietta
  orcidid: 0000-0003-3021-8919
  surname: Smulevich
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  organization: Dipartimento di Chimica "Ugo Schiff", Università di Firenze , Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy
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Snippet The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a...
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StartPage 1887
SubjectTerms Animals
Carbon Monoxide - chemistry
Cardiolipins - chemistry
Cardiolipins - metabolism
Cloning, Molecular
Cytochromes c - chemistry
Cytochromes c - genetics
Cytochromes c - metabolism
Escherichia coli - genetics
Escherichia coli - metabolism
Gene Expression
Genes, Synthetic
Histidine - chemistry
Horses
Hydrogen Bonding
Methionine - chemistry
Myocardium - chemistry
Protein Binding
Protein Folding
Protein Structure, Secondary
Protein Unfolding
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Temperature
Title Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only
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