255 Gen. Physiol. Biophys. (2010), 29, 255–265 doi:10.4149/gpb_2010_03_255 Rate of oxidative modifcation of cytochrome c by hydrogen peroxide is modulated by Hofmeister anions Nataša Tomášková 1 , Lenka Varinská 1, 2 and Erik Sedlák 1 1 Department of Biochemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University, Košice, Slovakia 2 Department of Pharmacology, Faculty of Medicine, P. J. Šafárik University, Košice, Slovakia Abstract. Cytochrome c (cyt c) and other heme proteins are oxidatively modifed in the presence of hydrogen peroxide in a concentration- and time-dependent manner. Cyt c modifcation has been monitored by several spectral probes by absorption spectroscopy (at wavelengths 410 nm, 530 nm), and circular dichroism (222, 268, 288 and 417 nm). Kinetics monitored with these spectral probes indicates that the oxidative modifcation of cyt c: i) proceeds in the order: heme → aromatic amino acids → secondary structure, and ii) the rate of the oxidative modifcation is proportional to the protein fexibility. Te fexibility of cyt c was modulated by anions of Hofmeister series (sulfate, chloride, perchlorate) (Varhač et al. 2009). A minimalist scheme of the interaction of cyt c with hy- drogen peroxide can be described by two steps: 1) interaction of hydrogen peroxide with heme iron forming the postulated ferryl intermediate, 2a) oxidation of another molecule of hydrogen peroxide and 2b) parallel oxidation of close amino acid residue(s) and/or heme. Te catalase activity of cyt c is independent from the presence of Hofmeister anions, which indicates that both steps (1 and 2a) in the catalase reaction are independent from the fexibility of the heme region of the protein matrix. On the other hand, the fexibility of the polypeptide chain of the protein modulates the rate of parallel oxidative modifcation of the heme and amino acid residues. Key words: Protein fexibility — Protein dynamics — Protein stability — Oxidative damage — Heme proteins Correspondence to: Erik Sedlák, Department of Biochemistry, Institute of Chemistry, Faculty of Science, P. J. Šafárik University, Košice, Slovakia E-mail: erik.sedlak@upjs.sk Introduction Cytochrome c (cyt c), a globular protein which contains a heme prosthetic group, is a typical example of multifunc- tional protein with two important physiological roles: i) to mediate electron shuttling between ubiquinol-cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV) during mitochondrial respiration, and ii) to serve as a factor regulating preapoptotic events (Liu et al. 1996; Acehan et al. 2002). Moreover, this cyt c is able to catalyze peroxidase-like reactions in the presence of an electron acceptor, such as hydrogen peroxide or an organic hydroperoxide (Radi et al. 1991; Vazquez-Duhalt 1999; Ka- gan et al. 2005). In addition, cyt c is known to alter both the generation and elimination of hydrogen peroxide (Deterding et al. 1998; Zhao et al. 2003), and to regenerate dioxygen from superoxide radical anion (Pereverzev et al. 2003). In classical peroxidase reaction the oxidation of substrates generally proceeds through two oxo-ferryl intermediates, called Compound I and Compound II, which are two and one oxidizing equivalents, respectively, above the Fe(III) state (Dawson 1988). Hemoglobin (Smith and Beck 1967), my- oglobin (Kelman et al. 1994), cyt c (Vazquez-Duhalt 1999), heme peptide microperoxidase (Casella et al. 2000) and heme c (Shedbalkar et al. 1988) show peroxidase-like activ- ity similar to classical peroxidases, though their reactivities with hydrogen peroxide are quite diferent. Although cyt c is a sluggish catalase/peroxidase, it can be physiologically relevant due to its high in vivo concentration (Belikova et al. 2006). Cyt c is located in the intermembrane mitochondrial space, where it is exposed to hydrogen peroxide released by mitochondria. All heme proteins including peroxidases are sensitive to oxidation by hydrogen peroxide. Te oxidative inactivation of heme proteins seems to be mechanism-