Biochemical Engineering Journal 28 (2006) 156–160 Peroxidative catalytic behavior of cytochrome c solubilized in reverse micelles Tsutomu Ono a,∗ , Masahiro Goto b a Department of Chemical Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan b Department of Applied Chemistry, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan Received 7 July 2005; received in revised form 12 October 2005; accepted 24 October 2005 Abstract Solubilization of horse heart cytochrome c into reverse micelles, a self-assembled nanostructure composed of sodium di-2-ethylhexyl sulfos- uccinate (AOT), enhances the peroxidase activity in the presence of hydrogen peroxide. The catalytic activity of cytochrome c hosted in reverse micellar solution is 10-fold higher than that in water, and which depends on Wo, a molar ratio of water to AOT, and pH in aqueous droplets. In addition, the fluorescence intensity based on the tryptophan residue and the visible absorption identifying the axial Met 80-Fe bond imply that the reverse micellar solubilization induces the cleavage of the heme crevice and thus enables the formation of peroxidase-like peroxide-heme complexes. These results suggest that cytochrome c solubilized in reverse micelles provides a homogeneous catalyst as peroxidase available in organic media. © 2005 Elsevier B.V. All rights reserved. Keywords: Microemulsion; Peroxidase; Surfactant; Oxidation 1. Introduction It has been known that cytochrome c functions in the elec- tron transfer on the inner mitochondrial membrane. Such a membrane-associated protein interacts with membrane sur- face, and their structural and physiological properties are often altered. Several studies suggested that this structural change could regulate the electron transfer from dehydrogenase systems to cytochrome c oxidase prior to reduction of molecular oxygen [1–3]. Indeed, binding of cytochrome c to negatively charged lipid involved a disruption of the native compact structure and partially unfolding on the membrane. Circular dichroism (CD) spectra, tryptophan fluorescence and heme absorbance revealed that cytochrome c bound to lipid membrane disrupted packing of core side chains and heme crevice, but retained a highly - helical structure [2]. The disruption of cytochrome c conformation leads to the fluctuation of its catalytic activity. Salamon and Tollin exhibited that the interaction of cytochrome c with lipid bilayer membrane brought about a positive shift of the cytochrome c redox poten- ∗ Corresponding author. Tel.: +81 92 802 2768; fax: +81 92 802 2768. E-mail address: tono@chem-eng.kyushu-u.ac.jp (T. Ono). tial and resulted in a large decrease in the electron transfer rate [4]. Apart from an electron transfer, a bilayer-bound cytochrome c showed peroxidase and N-demethylase activity, and which is also induced by structural perturbation based on the electro- static interaction between synthetic anionic amphiphiles and the cationic charged domain of cytochrome c [5–7]. Peroxidases catalyze one-electron oxidation of a wide variety of organic substrates through activation by hydrogen peroxide. Peroxidase available in organic media, therefore, is a promising biocatalyst for specific organic syntheses and bioremediation technology of harmful lipophilic molecules. Recently, oxidative polymerization of phenol, aniline, and their derivatives catalyzed by peroxidase in nonaqueous media has been noted as a new methodology to make environment-friendly resins [8], and to produce a chiral organic molecule through regio- and enantios- elective oxidation [9]. Most substrates (or monomers) and their oxidative products are hydrophobic and less soluble in aqueous solutions. In contrast, peroxidase is insoluble and less active in an organic solvent. Therefore, improving peroxidase activ- ity in organic media is most attractive for biotransformation of hydrophobic substrates: e.g. polyphenol with a high molec- ular weight [10–13], fluorescent naphthol-based polymer [14] and conducting polyaniline [15], and oxidation of organo-sulfur compounds [16]. In particular, a peroxidase-mediated polyphe- 1369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2005.10.005