Role of lysines in cytochrome c-cardiolipin interaction.

Biochemistry (2013-06-07)
Federica Sinibaldi, Barry D Howes, Enrica Droghetti, Fabio Polticelli, Maria Cristina Piro, Donato Di Pierro, Laura Fiorucci, Massimo Coletta, Giulietta Smulevich, Roberto Santucci
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cytochrome c from equine heart, ≥95% (SDS-PAGE)
Sigma-Aldrich
L-Lysine, ≥98% (TLC)
Sigma-Aldrich
L-Lysine monohydrochloride, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
Cytochrome c from equine heart, ≥95% based on Mol. Wt. 12,384 basis
Sigma-Aldrich
Cytochrome c from bovine heart, ≥95% based on Mol. Wt. 12,327 basis
Sigma-Aldrich
L-Lysine monohydrochloride, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
Cytochrome c from bovine heart, ≥95% based on Mol. Wt. 12,327 basis
Sigma-Aldrich
L-Lysine, crystallized, ≥98.0% (NT)
Supelco
L-Lysine monohydrochloride, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
L-Lysine monohydrochloride, BioUltra, ≥99.5% (AT)
Sigma-Aldrich
Cytochrome c from equine heart, BioUltra, ≥99% (SDS-PAGE)
Sigma-Aldrich
Cytochrome c from equine heart, suitable for GFC marker, BioReagent
Supelco
L-Lysine monohydrochloride, certified reference material, TraceCERT®
Sigma-Aldrich
Cytochrome c from Saccharomyces cerevisiae, ≥85% based on Mol. Wt. 12,588 basis
Sigma-Aldrich
ProteoMass Cytochrome c MALDI-MS Standard, vial of 10 nmol, (M+H+) 12,361.96 Da by calculation
Sigma-Aldrich
L-Lysine acetate salt, ≥98% (HPLC)
Supelco
L-Lysine Acetate, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Cytochrome c from pigeon breast muscle, ≥95% based on Mol. Wt. 12,173 basis