Chemistry and Physics of Lipids 163 (2010) 538–544 Contents lists available at ScienceDirect Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip Interaction between non-anionic phospholipids and cytochrome c induced by reactive oxygen species Nazha Sidahmed-Adrar a , Catherine Marchetti a , Dominique Bonnefont-Rousselot b , Juliette Thariat c , Delphine Onidas a , Daniel Jore a , Monique Gardes-Albert a , Fabrice Collin a,∗ a Equipe de Physico-Chimie des Espèces Radicalaires, CNRS UMR 8601, Université Paris Descartes, 45 rue des Saints-Pères, 75006 Paris, France b Laboratoire de Biochimie Métabolique et Clinique, EA 3617, Faculté de Pharmacie Paris Descartes, 4 avenue de l’Observatoire, 75006 Paris, France c Département de Radiothérapie, Centre Anti-Cancer Antoine Lacassagne, 33 avenue Valombrose, 06189 Nice Cedex 2, France article info Article history: Received 26 November 2009 Received in revised form 16 March 2010 Accepted 6 April 2010 Available online 14 April 2010 Keywords: Cytochrome c Phospholipid Reactive oxygen species Peroxidation Mass spectrometry abstract The oxidative interaction of cytochrome c (Cyt c) with liposomes of Palmitoyl Linoleyl Phosphatidyl Choline (PLPC) initiated by radio-induced free radicals was investigated. Results showed that the perox- idation of PLPC is decreased in the presence of Cyt c, meaning that this latter is the preferential target of hydroxyl radicals. In addition, when Cyt c was incubated with peroxidized PLPC, it was found to be able to decompose hydroperoxides of PLPC into hydroxides. The peroxidase activity of Cyt c proceeded via the opening of the tertiary structure of Cyt c, as suggested by the loss of the sixth coordination bond of the heme-iron. Even if it is known to preferentially interact with cardiolipin, this work shows that Cyt c is also able to interact with hydroperoxide species of non-anionic phospholipids. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Phospholipids play an important role in cellular processes by organising themselves in a membrane bilayer, which forms, together with intrinsic proteins, a permeable barrier of cells and organelles (Dowhan, 1997). Phosphatidylcholines, a heterogeneous group whose fatty-acyl chains vary in length and degree of unsat- uration, are one of the major components of eukaryotic biological membranes. Most naturally occurring phosphatidylcholines con- tain polyunsaturated fatty-acyl moieties which are particularly susceptible to oxidation mediated by free radicals (Stark, 1991). Their oxidation leads to hydroperoxides as primary products (Kim and Labella, 1987) and is usually responsible for breakdown or mal- function of membranes (Pacifici et al., 1994; Antunes et al., 1996). Such radical chemical reactions have been considerably studied (Halliwell and Gutteridge, 1999). Cytochrome c (Cyt c) is located on the outer surface of the inner mitochondrial membrane. It is a 12 kDa (in mammals, 104 amino acid residues) globular protein with a heme group (protoporphyrin IX and Fe 3+ ) covalently bound to two cystein residues (Cys-14 and ∗ Corresponding author. Present address: Laboratoire des Mécanismes Réac- tionnels (CNRS UMR 7651), Ecole Polytechnique, 91128 Palaiseau Cedex, France. Tel.: +33 1 42 86 21 72; fax: +33 1 69 33 48 23. E-mail address: fabrice.collin@parisdescartes.fr (F. Collin). Cys-17) and coordinately bound to His-18 and Met-80. If Cyt c is able to react and exhibit a peroxidase activity towards cardi- olipins, it cannot interact with positively charged phospholipids (Belikova et al., 2006). However, it is known to react with hydroper- oxides in a way that implies production of intermediate radical species (Cadenas et al., 1980; Barr and Mason, 1995). Recently, Cyt c has been shown to trigger the decomposition of -linolenic acid hydroperoxide by causing a homolytic scission and releasing singlet oxygen (Sun et al., 2007). However, the behaviour of Cyt c has never been investigated in decomposing polyunsaturated fatty-acyl chain hydroperoxides from phospholipid, and in particular hydroperox- ides from phosphocholine. This latter is known not to interact with Cyt c (Belikova et al., 2006), but direct observation was done regard- ing Cyt c ability to cross neutral lipid membranes in a recent work (El Kirat and Morandat, 2009). Moreover, during oxidative stress, both cytochrome c and phospholipids are potential targets of reac- tive oxygen species (ROS), such as hydroxyl and superoxide free radicals. In a previous study, dedicated to the oxidation of Cyt c mediated by HO • and/or O 2 •- free radicals, protoporphyrin IX was found to be oxidized along with several residues of the polypeptidic chain of Cyt c that where mainly hydroxylated or carbonylated (Val- 11, Glu-12, His-33, Phe-36, Pro-76, Gly-77, Thr-78, Lys-79, Met-80, Lys-86) (Thariat et al., 2008). Among them, Met-80 appeared as the major target for ROS and was oxidized into methionine sulfoxide. The resulting disruption of the Met-80/heminic iron coordination bond is able to initiate the conversion of Cyt c from an electron car- 0009-3084/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2010.04.002