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Theoretical Study of the Catalytic Mechanism of Catechol Oxidase

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Theoretical Study of the Catalytic Mechanism of Catechol Oxidase J Biol Inorg Chem (2007) 12:1251–1264 DOI 10.1007/s00775-007-0293-z ORIGINAL PAPER Theoretical study of the catalytic mechanism of catechol oxidase Mireia Gu¨ell Æ Per E. M. Siegbahn Received: 20 June 2007 / Accepted: 16 August 2007 / Published online: 20 September 2007 Ó SBIC 2007 Abstract The mechanism for the oxidation of catechol Introduction by catechol oxidase has been studied using B3LYP hybrid density functional theory. On the basis of the X-ray Proteins containing copper ions at their active sites are structure of the enzyme, the molecular system investigated usually involved as redox catalysts in a wide range of includes the first-shell protein ligands of the two metal biological processes. Type-3 active-site copper proteins centers as well as the second-shell ligand Cys92. The cycle contain a dicopper core in which both copper ions are starts out with the oxidized, open-shell singlet complex surrounded by three nitrogen donor atoms from histidine 2 2 with oxidation states Cu2(II,II) with a l-g :g bridging residues [1, 2]. A characteristic feature of the proteins with peroxide, as suggested experimentally, which is obtained this active site is their ability to reversibly bind dioxygen at from the oxidation of Cu2(I,I) by dioxygen. The substrate ambient conditions. The Cu(II) ions in the oxy state of of each half-reaction is a catechol molecule approaching these proteins are strongly antiferromagnetically coupled, the dicopper complex: the first half-reaction involves Cu(I) leading to electron paramagnetic resonance (EPR) silent oxidation by peroxide and the second one Cu(II) reduction. behavior. This class is represented by three proteins: The quantitative potential energy profile of the reaction is hemocyanin, catechol oxidase and tyrosinase. discussed in connection with experimental data. Since no Proteins with type-3 copper centers can serve either as protons leave or enter the active site during the catalytic oxygenase/oxidase enzymes or as dioxygen transport pro- cycle, no external base is required. Unlike the previous teins [2]. An example of an oxygen carrier is hemocyanin. density functional theory study, the dicopper complex has a Hemocyanins can be divided into two classes depending on charge of +2. their biological source: the arthropodan and the molluscan hemocyanins [3–5]. Keywords Catechol oxidase Á Copper enzymes Á Catechol oxidase, which is also known as o-diphenol O2 cleavage Á Hybrid density functional theory oxidase, catalyzes exclusively the oxidation of catechols (i.e., o-diphenols) to the corresponding o-quinones (called catecholase activity) (Fig. 1)[6]. The resulting highly reactive quinones autopolymerize to form brown poly- phenolic catechol melanins, a process thought to protect M. Gu¨ell (&) damaged plants from pathogens or insects [7]. The rate for Institut de Quı´mica Computacional, catechol conversion in sweet potatoes has been measured Universitat de Girona, to be 2.3 · 103/s [8], corresponding to a rate-limiting free- Campus de Montilivi, 17071 Girona, Spain energy barrier of around 13 kcal/mol. e-mail: [email protected] In contrast to catechol oxidase, the strongly related tryrosinase shows additional monooxygenase activity P. E. M. Siegbahn (Fig. 1). This so-called cresolase activity enables the Department of Biochemistry and Biophysics, Stockholm University, enzyme to accept also monophenols (like tyrosine and 106 91 Stockholm, Sweden cresol). Catechol oxidases are found in plant tissues and in 123 1252 J Biol Inorg Chem (2007) 12:1251–1264 some insects and crustaceans, whereas tryrosinases can be isolated from a broader variety of plants, fungi, bacteria, mammalians, crustaceans and insects [2]. The differentia- tion between catechol oxidase and tryrosinase is not rigorous, as some catechol oxidases also show weak monooxygenase activity. However, these catechol oxidases often do not accept tyrosine as a substrate [9, 10]. Klabunde et al. [11] isolated the first crystal structures of the catechol oxidase from Ipomoea batatas (sweet potato) in three catalytic states: the native met [Cu(II)Cu(II)] state, the reduced deoxy [Cu(I)Cu(I)] form, and in the complex with the inhibitor phenylthiourea. This enzyme was found to be monomeric and ellipsoidal in shape (Fig. 2). Its secondary structure is primarily a-helical with the core of Fig. 2 Tertiary structure of the oxidized catechol oxidase (PDB code the enzyme formed by a four-helix bundle. The helical 1BT3) [11]. Cu(II) ions are given as yellow spheres and important active-site residues are shown. The picture was generated using the bundle accommodates the catalytic dinuclear copper cen- VMD 1.8.5 molecular visualization program. See Fig. 3 for a more ter, where each of the two copper ions is coordinated by detailed picture of the active site three histidine residues. One of the key features of the catechol oxidase active site is an unusual thioether bridge sulfur atom of phenylthiourea is coordinated to both Cu(II) between Cys92 and His109, one of the ligands of CuA centers, increasing the distance between them to 4.2 A˚ . The (Fig. 3). Apart from the geometrical constraints added to amide nitrogen interacts weakly with the CuB center (Cu–N the CuA site, no function of the chemistry performed by the distance of 2.6 A˚ ), completing its square-pyramidal enzyme has been ascribed to this covalent bridge. geometry. In the native met state, the two copper ions are 2.9 A˚ The oxy form of catechol oxidase can be obtained by apart. In addition to six histidine residues, a bridging sol- treating the met form of the enzyme with hydrogen per- vent molecule, most likely hydroxide anion, was refined in oxide [12]. close proximity to the two metal centers (CuA–O 1.9 A˚ , As previously mentioned, catechol oxidase catalyzes the CuB–O 1.8 A˚ ), completing the coordination sphere of the oxidation of catechols to the respective quinones through a copper ions to a trigonal pyramid. EPR data reveal a strong four-electron reduction of dioxygen to water. Krebs and antiferromagnetic coupling between the copper ions, in line coworkers proposed a mechanism for the catalytic process, with a solvent molecule bridging two metal centers, as based on biochemical and spectroscopic data [2, 12, 13], as found in the crystal structure. well as structural data [11], which is depicted in Fig. 4 Upon reduction of the Cu(II) ions to the +1 oxidation [14]. The catalytic cycle begins with the met form of cat- state, the distance between them increases to 4.4 A˚ , while echol oxidase, which is the resting form of the enzyme. The the histidine residues move only slightly, and no significant dicopper(II) center of the met form reacts with 1 equivalent change was observed for other residues of the protein [11]. of catechol, leading to the formation of quinone and to the On the basis of the residual electron density maps, a water reduced deoxy dicopper(I) state. This step is supported by molecule was positioned at a distance of 2.2 A˚ from the the observation that stoichiometric amounts of the quinone CuA atom. Thus, the coordination sphere around the CuA product form immediately after the addition of catechol, ion is a distorted trigonal pyramid, with three nitrogen even in the absence of dioxygen [11, 14]. On the basis of atoms from the histidine residues forming a basal plane, the structure of catechol oxidase with the bound inhibitor while the coordination sphere around the CuB ion is best phenylthiourea, the monodentate binding of the substrate to described as square planar with one missing coordination the CuB center has been proposed. Afterwards, dioxygen site. binds to the dicopper(I) active site, replacing the solvent When phenylthiourea binds to catechol oxidase, it molecule bonded to CuA in the reduced enzyme. UV–vis replaces the hydroxo bridge, present in the met form. The spectroscopy and Raman data suggested that dioxygen Fig. 1 Reaction pathway of the 1/2 O2 1/2 O2 H2O oxygenation and oxidation OH OH O catalyzed by tyrosinase and catechol oxidase cresolase activity catecholase activity OH O (tyrosinase) (tyrosinase and catechol oxidase) 123 J Biol Inorg Chem (2007) 12:1251–1264 1253 Fig. 3 Active site of the oxidized catechol oxidase (PDB code 1BT3) [11] binds in a bridging side-on l-g2:g2 binding mode with a tetragonal planar coordination by His240, His244 and the copper–copper separation of 3.8 A˚ , as determined by dioxygen molecule in the basal plane. The CuA site retains extended X-ray absorption fine structure spectroscopy for the tetragonal pyramidal geometry with dioxygen, His88 the oxy state [12]. The observed binding mode of phenyl- and His118 in the equatorial positions, His109 in an axial thiourea and the modeled catechol-binding mode suggest position and a vacant sixth coordination site. In this pro- 2– that simultaneous binding of catechol and dioxygen is posed ternary catechol oxidase–O2 –catechol complex, two possible. In this model, CuB is six-coordinated with a electrons can be transferred from the substrate to the Fig. 4 Catalytic cycle of O OH catechol oxidase as proposed H O + by Klabunde et al. [11] 2 O His His OH His CuIIA BCuII His O His H His H+ met state 2H+ HO HO O O His His His His O His CuIIA BCuII His His CuIIA B CuII His O His O His H His His oxy state + H2O + H His OH2 His O I I OH His Cu A BCu His O2 + His His O OH deoxy state 123 1254 J Biol Inorg Chem (2007) 12:1251–1264 peroxide, followed by cleavage of the O–O bond, loss of the substrate, participate as proton storage/delivery devi- water and the formation of the quinone product, together ces.
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