title: Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only.
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.
authors.affiliation: Dipartimento di Chimica "Ugo Schiff", Università di Firenze , Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy.
authors.forename: Lisa
authors.initials: L
authors.lastname: Milazzo
authors.affiliation: Dipartimento di Chimica "Ugo Schiff", Università di Firenze , Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Tognaccini
authors.affiliation: Dipartimento di Chimica "Ugo Schiff", Università di Firenze , Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy.
authors.forename: Barry D
authors.initials: BD
authors.lastname: Howes
authors.affiliation: Dipartimento di Medicina Sperimentale e Chirurgia, Università di Roma "Tor Vergata" , Via Montpellier 1, 00133 Rome, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Sinibaldi
authors.affiliation: Dipartimento di Medicina Sperimentale e Chirurgia, Università di Roma "Tor Vergata" , Via Montpellier 1, 00133 Rome, Italy.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Piro
authors.affiliation: Dipartimento di Fisica ed Astronomia, Università di Firenze , Via Sansone 1, 50019 Sesto Fiorentino (FI), Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Fittipaldi
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena , Via Aldo Moro 2, 53100 Siena, Italy.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Baratto
authors.affiliation: Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena , Via Aldo Moro 2, 53100 Siena, Italy.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Pogni
authors.affiliation: Dipartimento di Scienze Cliniche e Medicina Traslazionale, Università di Roma "Tor Vergata" , Via Montpellier 1, 00133 Rome, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Santucci
authors.affiliation: Dipartimento di Chimica "Ugo Schiff", Università di Firenze , Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy.
authors.forename: Giulietta
authors.identifier: 0000-0003-3021-8919
authors.initials: G
authors.lastname: Smulevich
mesh_terms: D000818:Animals; D002248:Carbon Monoxide / Q000737:chemistry*; D002308:Cardiolipins / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D006639:Histidine / Q000737:chemistry*; D006736:Horses; D006860:Hydrogen Bonding; D008715:Methionine / Q000737:chemistry*; D009206:Myocardium / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D002308:Cardiolipins; D011994:Recombinant Proteins; D006639:Histidine; D002248:Carbon Monoxide; D045304:Cytochromes c; D008715:Methionine