X-ray absorption analysis of the active site of Streptomyces antibioticus Tyrosinase upon binding of transition state analogue inhibitors Luigi Bubacco a,c, * , Roberto Spinazze a , Stefano della Longa b,c , Maurizio Benfatto c a Department of Biology, University of Padova, Via Ugo Bassi 58B, 35121, Padova, Italy b Department of Medicine, University of L’Aquila, L’Aquila, Italy c Laboratori Nazionali di Frascati dell’INFN – INFN, Frascati, Italy Received 12 February 2007, and in revised form 9 July 2007 Available online 17 July 2007 Abstract The key structural features that define the reaction mechanism of the binuclear copper enzyme Tyrosinase (Ty) from Streptomyces antibioticus were investigated by X-ray absorption spectroscopy. The data for the met form, the halide bound derivative and the adduct with the competitive inhibitor and transition state analogue Kojic acid were analysed using the recently developed MXAN package. This analysis permitted the definition of structural clusters that include all atoms within 5 A ˚ from the metal ions of the active site. The data obtained for the different forms provide validation of the structural models previously proposed on the basis of the magnetic properties investigated by both pulsed EPR and paramagnetic NMR spectroscopies. The structural model of the reaction center obtained in this solution study is compared with the crystallographic structures recently proposed for several derivatives of bacterial Ty to suggest that only one of these structures is relevant to solution conditions. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Tyrosinase; Streptomyces; X-ray absorption spectroscopy; Competitive inhibitor; Kojic acid; MXAN; Reaction center; Crystal structure The reaction mechanism of the binuclear copper enzyme Tyrosinase (Ty) (EC 1.14.18.1) is still object of debate. This enzyme, like catechol oxidase (CO) and the respiratory pig- ment hemocyanin (Hc), is characterised by the presence of a so-called ‘‘type-3 Cu’’ active site [1]. This early classifica- tion was based on the common spectroscopic properties of the binuclear copper center. In spite of both the importance of Ty and numerous attempts, only very recently crystal structures were obtained for several forms of the bacterial enzyme [2]. These structures refer to apo and to holo forms of the proteins in the different oxidation states accessible to the dinuclear center. These are the fully reduced [Cu(I) - Cu(I)] deoxy state that, upon reversible binding of molecu- lar oxygen, turns into the oxy form [Cu(II) O 2 2 Cu(II)] and into the fully oxidised met form [Cu(II) OH  Cu(II)]. Other relevant crystal structures are those of the deoxygen- ated form of Panulirus interruptus Hc [3], the deoxy, oxy and met forms of subunit II from Limulus polyphemus Hc [4,5], the oxy form of the odg domain of Octopus dofleini Hc [6] and the deoxy, met and inhibitor-bound forms of the Ipomoea batatas CO [7]. The definition of both the reaction mechanism and the related structural features requires direct structural infor- mation on all accessible forms of Ty in solution. The first approach was the generation of a paramagnetic center in the active site of bacterial Ty through partial reduction of the met form leading to the half-met form [Cu(I)–Cu(II)] [8,9]. Recently, a pulsed EPR study on the bacterial Ty per- mitted a detailed description of the coordination geometry of the single oxidised copper in the binuclear center and the detection of the protons bound to the oxygen atom of a water molecule directly coordinated to the copper ion. 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.07.008 * Corresponding author. Address: Department of Biology, University of Padova, Via Ugo Bassi 58B, 35121, Padova, Italy. Fax: +39 0498276300. E-mail address: luigi.bubacco@unipd.it (L. Bubacco). www.elsevier.com/locate/yabbi Archives of Biochemistry and Biophysics 465 (2007) 320–327 ABB