ORIGINAL PAPER Versatility of non-native forms of human cytochrome c: pH and micellar concentration dependence Matthieu Simon • Vale ´rie Metzinger-Le Meuth • Soizic Chevance • Olivier Delalande • Arnaud Bondon Received: 29 June 2012 / Accepted: 30 September 2012 / Published online: 16 October 2012 Ó SBIC 2012 Abstract In addition to its electron transfer activity, cyto- chrome c is now known to trigger apoptosis via peroxidase activity. This new function is related to a structural modifi- cation of the cytochrome upon association with anionic lipids, particularly cardiolipin present in the mitochondrial mem- brane. However, the exact nature of the non-native state induced by this interaction remains an active subject of debate. In this work, using human cytochromes c (native and two single-histidine mutants and the corresponding double mutant) and micelles as a hydrophobic medium, we succeeded, through UV–visible spectroscopy, circular dichroism spectroscopy and NMR spectroscopy, in fully characterizing the nature of the sixth ligand replacing the native methionine. Furthermore, careful pH titrations per- mitted the identification of the amino acids involved in the iron binding over a range of pH values. Replacement of the methionine by lysine was only observed at pH above 8.5, whereas histidine binding is dependent on both pH and micelle concentration. The pH variation range for histidine proton- ation is relatively narrow and is consistent with the mito- chondrial intermembrane pH changes occurring during apoptosis. These results allow us to rule out lysine as the sixth ligand at pH values close to neutrality and reinforce the role of histidines (preferentially His33 vs. His26) as the main can- didate to replace methionine in the non-native cyto- chrome c. Finally, on the basis of these results and molecular dynamics simulations, we propose a 3D model for non-native cytochrome c in a micellar environment. Keywords Human cytochrome c Á Micelles Á Sodium dodecyl sulphate Á NMR Á Paramagnetic NMR Introduction Cytochrome c is a soluble, small and basic haemoprotein that is located in the mitochondrial intermembrane space. Its historic function is to shuttle electrons from cytochrome c reductase to cytochrome c oxidase in the respiratory chain. In the native state, His18 and Met80 are the two axial ligands forming the iron hexacoordinated low-spin state (LS HM ). However, cyto- chrome c has been shown to be deeply involved in the early steps of apoptosis [1]. Electrostatic and hydrophobic interac- tions with cardiolipin, an exclusive mitochondrial phospho- lipid, are at the origin of the destabilization of the haem Electronic supplementary material The online version of this article (doi:10.1007/s00775-012-0946-4) contains supplementary material, which is available to authorized users. M. Simon Á V. Metzinger-Le Meuth Á S. Chevance Á O. Delalande Á A. Bondon (&) Universite ´ de Rennes 1 and UMR CNRS 6290, SIM, PRISM, Biosit, CS 34317, Campus de Villejean, 35043 Rennes Cedex, France e-mail: arnaud.bondon@univ-rennes1.fr Present Address: V. Metzinger-Le Meuth Universite ´ Paris 13, UFR SMBH, 74 rue Marcel Cochin, 93017 Bobigny, France V. Metzinger-Le Meuth Faculte ´ de Pharmacie et Me ´decine, INSERM U1088, MP3C, Rue des Louvels, 80037 Amiens, France Present Address: S. Chevance Universite ´ de Rennes 1 and UMR CNRS 6226, ICMV, Campus de Beaulieu, 35042 Rennes Cedex, France 123 J Biol Inorg Chem (2013) 18:27–38 DOI 10.1007/s00775-012-0946-4