A new format of electrodes for the electrochemical reduction of cytochromes P450 Victoria V. Shumyantseva a, * , Tatiana V. Bulko a , Natalia F. Samenkova a , Galina P. Kuznetsova a , Sergei A. Usanov b , Holger Schulze c , Till T. Bachmann c , Rolf D. Schmid c , Alexander I. Archakov a a Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya Street, 10, Moscow 119121, Russia b Institute of Bioorganic Chemistry, National Academy of Science of Belarus, Kuprevicha 5, Minsk 220141, Belarus c Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany Received 23 September 2005; received in revised form 20 March 2006; accepted 21 March 2006 Available online 25 April 2006 Abstract New approach to the electrochemical reduction of cytochromes P450 (P450s, CYPs) at electrodes chemically modified with appropri- ate substrates for P450s (‘‘reverse’’ electrodes) was proposed. The method is based on the analysis of cyclic voltammograms, square-wave voltammograms and amperograms with subsequent determination of electrochemical characteristics such as catalytic current and redox potential. The sensitivity of proposed method is 0.2–1 nmol P450/electrode. The changes of maximal current and of redox potentials in square-wave voltammograms as well as the changes of catalytic current in amperometric experiments proved to be informative and reliable. Planar regime of screen-printed electrodes (strip-type sensors) enabled to utilise 20–60 ll of electrolyte volume. The enzyme– substrate pairs P450 2B4/benzphetamine and P450scc/cholesterol were investigated. Electrochemical parameters of electrodes with unspecific P450 substrates differed considerably from electrodes with appropriate substrates. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450; Electrochemistry; Electrocatalysis; Cyclic voltammetry; Square-wave voltammetry 1. Introduction Cytochromes P450 (P450s) belong to a multigene family of heme proteins which catalyse the NADPH-dependent monooxygenation and other reactions of different exobi- otic and endobiotic lipophilic substrates. These enzymes are unique in being able to hydroxylate non-activated car- bon atoms (CAH bonds) [1]. Products of substrates’ metabolism usually serve as regulators in cells or are excreted from organisms. Cytochromes are capable of met- abolising over 1,000,000 chemicals and involve about 60 distinct classes of biotransformation reactions, i.e. the reac- tions of hydroxylation, N-, O- or S-demethylation, dealky- lation, epoxidation and others [2]. This specific feature of P450s makes them highly promising for the use in pharma- ceutical drug assays, in stereo-directed synthesis of steroids and as markers of diseases [3]. The efficiency improvement of hemoproteins-catalysed reactions is an important practi- cal task [3]. The biological relevance of cytochrome P450 determination is demonstrated in following studies [4,5]. Electron transfer in biological systems occurs in the presence of electron donors such as NADPH or NADH and is generally effected by electron transfer mediators, such as flavin nucleotides. During reduction NADPH or NADH are exhausted. One of the alternative methods of P450s reduction is electrochemical reduction with an elec- trode as electron source [6,7]. Use of electrochemical sys- tems in the redox enzymes’ reactions, particularly in metalloenzymes’ reactions, is finding an increasing applica- tion. P450s and enzyme electrodes based on P450s are 0162-0134/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2006.03.007 * Corresponding author. Tel.: +7 495 246 58 20; fax: +7 495 245 08 57. E-mail address: viktoria.shumyantseva@ibmc.msk.ru (V.V. Shumyant- seva). www.elsevier.com/locate/jinorgbio Journal of Inorganic Biochemistry 100 (2006) 1353–1357 JOURNAL OF Inorganic Biochemistry