title: Role of lysines in cytochrome c-cardiolipin interaction.
abstract: Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c-cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c-cardiolipin interaction, as these side chains appear to be critical for cytochrome c-cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet-visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c-cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.
authors.affiliation: Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, 00133 Rome, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Sinibaldi
authors.forename: Barry D
authors.initials: BD
authors.lastname: Howes
authors.forename: Enrica
authors.initials: E
authors.lastname: Droghetti
authors.forename: Fabio
authors.initials: F
authors.lastname: Polticelli
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Piro
authors.forename: Donato
authors.initials: D
authors.lastname: Di Pierro
authors.forename: Laura
authors.initials: L
authors.lastname: Fiorucci
authors.forename: Massimo
authors.initials: M
authors.lastname: Coletta
authors.forename: Giulietta
authors.initials: G
authors.lastname: Smulevich
authors.forename: Roberto
authors.initials: R
authors.lastname: Santucci
mesh_terms: D000818:Animals; D017209:Apoptosis; D002308:Cardiolipins / Q000737:chemistry / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D009206:Myocardium / Q000201:enzymology*; D009195:Peroxidase / Q000737:chemistry; D011485:Protein Binding; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary
chemical_list: D002308:Cardiolipins; D045304:Cytochromes c; D009195:Peroxidase; D008239:Lysine