Review Spectroscopic characterization of cytochrome P450 Compound I Christiane Jung a,⇑ , Simon de Vries b , Volker Schünemann c,⇑ a Max-Delbrück-Center for Molecular Medicine, Robert-Rössle Strasse 10, 13125 Berlin, Germany b Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands c Dept. of Physics, University Kaiserslautern, Erwin-Schrödinger-Strasse 46, 67663 Kaiserslautern, Germany article info Article history: Available online 30 December 2010 Keywords: Cytochrome P450 Compound I Iron-oxo species Spectroscopy abstract The cytochrome P450 protein-bound porphyrin complex with the iron-coordinated active oxygen atom as Fe(IV)@O is called Compound I (Cpd I). Cpd I is the intermediate species proposed to hydroxylate directly the inert carbon–hydrogen bonds of P450 substrates. In the natural reaction cycle of cytochrome P450 Cpd I has not yet been detected, presumably because it is very short-lived. A great variety of exper- imental approaches has been applied to produce Cpd I artificially aiming to characterize its electronic structure with spectroscopic techniques. In spite of these attempts, none of the spectroscopic studies of the last decades proved capable of univocally identifying the electronic state of P450 Cpd I. Very recently, however, Rittle and Green [9] have shown that Cpd I of CYP119, the thermophillic P450 from Sulfolobus acidocaldarius, is univocally a Fe(IV)@O–porphyrin radical with the ferryl iron spin (S = 1) anti- ferromagnetically coupled to the porphyrin radical spin (S 0 = 1/2) yielding a S tot = 1/2 ground state very similar to Cpd I of chloroperoxidase from Caldariomyces fumago. In this mini-review the efforts to char- acterize Cpd I of cytochrome P450 by spectroscopic methods are summarized. Ó 2010 Elsevier Inc. All rights reserved. Introduction The name cytochrome P450 (P450) 1 stays for a large number of enzymes found in different organisms such as bacteria, fungi, mam- mals, and humans which catalyze the conversion of a variety of chemically diverse compounds [1]. Cytochromes P450 are heme thiolate proteins and members of the so-called CYP-superfamily cur- rently consisting of 12,476 already named P450s. Bioinformatic anal- yses suggest the existence of approximately 6000 additional members, which have not yet been named [2]. In cytochrome P450s, the heme is bound to the protein matrix by coordination of the H-bonded, negatively charged sulfur atom of a cysteine to the iron, by salt links, by several van der Waals contacts and by H-bonded water molecules present in the heme pocket [3] (Fig. 1). The main steps in the P450 reaction cycle (Fig. 2) include (i) substrate (–C–H) binding to the Fe 3+ form of P450 accompanied by a spin state change from low-spin to high-spin in most of the enzymes; (ii) first reduction to build up the Fe 2+ state; (iii) binding of O 2 to the Fe 2+ heme, (iv) delivery of the second electron; (v) cleavage of the O–O bond of the iron bound dioxygen; (vi) insertion of an oxygen atom into the substrate; and (vii) release of the hydroxylated product (–C–OH). Along the Step (v) an intermediate heme iron-bound oxygen species is formed that is called ‘‘Compound I’’ (Cpd I) and believed to represent the active species, which directly hydroxylates the substrates [4]. There has been a general consensus in the P450 community that Cpd I, resulting from the heterolytic splitting of the O–O bond, does really exist and represents an iron-oxo species with iron in the Fe(IV) state and the porphyrin with the cation radical in the p-system [5–8]. The addition of one electron to Cpd I results in the formation of a species which is called Compound II (Cpd II). The way to prove this Cpd I hypothesis is to compare the spec- troscopic properties and parameters of this presumed Cpd I species with well-studied reference samples for which the spectroscopic assignment to Fe(IV) and the porphyrin-p-cation radical has been unambiguously demonstrated. Although a number of experimental and theoretical studies have been performed in the last 45 years, the electronic structure of P450 Cpd I has not been finally charac- terized because this intermediate is very short-lived and difficult 0003-9861/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2010.12.029 ⇑ Corresponding authors. Present address: KKS Ultraschall AG, Medical Surface Center, Frauholzring 29, CH-6422 Steinen, SZ, Switzerland. Fax: +41 41 8104508 (C. Jung). E-mail addresses: christiane_jung@bluewin.ch, christiane_jung@hotmail.com, cjung@mdc-berlin.de (C. Jung), schuene@physik.uni-kl.de (V. Schünemann). 1 Abbreviations used: P450cam, cytochrome P450cam from Pseudomonas putida, CYP101; Pdx, putidaredoxin; BMP, the heme protein domain of the monooxygenase P450BM-3 from Bacillus megaterium, CYP102; HRP, horseradish peroxidase; CPO, chloroperoxidase from Caldariomyces fumago; NADP(H), nicotinamide adenine (di)nucleotide phosphate, reduced form; m-CPBA, meta-chloroperoxybenzoic acid; PA, peroxy acetic acid; EPR, electron spin paramagnetic resonance; EXAFS, extended X-ray absorption fine structure; ENDOR, electron-nuclear double resonance; DFT, density functional theory; NIS, nuclear inelastic scattering; IR, infrared; RR, resonance Raman; NOS, nitric oxide synthase; iNOSox, inducible NOS oxygen domain; nNOSox, neuronal NOS oxygen domain. Archives of Biochemistry and Biophysics 507 (2011) 44–55 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi