Insights into the nature of the hydrogen bonding of Å Tyr 272 in apo-galactose oxidase Laurent Benisvy a, * , Deborah Hammond b , David J. Parker b , E. Stephen Davies b , C. David Garner b, * , Jonathan McMaster b, * , Claire Wilson b , Frank Neese a,d, * , Eberhard Bothe a , Robert Bittl c , Christian Teutloff c a Max-Planck-Instut fu ¨ r Bioanorganische Chemie, Stiftstrasse 34-46, Mu ¨ lheim an der Ruhr, D45470, Germany b School of Chemistry, The University of Nottingham, University Park, Nottingham, NG7 2RD, UK c Institut fu ¨ r Experimentalphysik, Freie Universita ¨ t, Berlin, Arnimallee 14,14195 Berlin, Germany d Institut fu ¨ r Physikalische and Theoretische Chemie, Universita ¨ t Bonn, Wegelerstrasse 12, D-53115 Bonn, Germany Received 16 April 2007; received in revised form 3 July 2007; accepted 12 July 2007 Available online 21 July 2007 This article is in appreciation of the many significant scientific contributions of Edward Stiefel; an individual who brought inorganic chemistry to life – in both senses – and was an inspiration to many and a good friend over many years (to CDG). Abstract The synthesis and structure of an o-methylthio-phenol-imidazole, 2-(2 0 -(4 0 -tert-butyl-6 0 -methylsulfanyl)-hydroxyphenyl))-4,5-diphe- nyl-imidazole ( MeS LH), is reported; X-ray crystallographic studies have shown that MeS LH involves an O–HÆÆÆN + hydrogen bond between the phenol and an imidazole nitrogen. MeS LH undergoes a reversible, one-electron, oxidation to form the radical cation [ MeS LH] Å+ the EPR spectrum of which is remarkably similar to that of Å Tyr 272 in Cu-free, oxidized, apo-GO. Density Functional Theory calculations, have shown that the proton-transferred (R–O Å ÆÆÆH–N + ) form of [ MeS LH] Å+ has a spin density distribution – with a substan- tial delocalization of the unpaired electron spin density onto the ortho sulfur atom – and EPR properties that are in good agreement with those of Å Tyr 272 in Cu-free, oxidized, apo-GO whereas the non-proton-transferred (R–O Å+ –HÆÆÆN) form does not. The results reported herein are a further demonstration of the influence of hydrogen bonding on the nature and properties of phenoxyl radicals and strongly suggest that the phenoxyl oxygen of Å Tyr 272 in Cu-free, oxidized, apo-GO is involved in a O Å ÆÆÆH–O/N hydrogen bond. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Oxidized apo-galactose oxidase; Density functional calculations; Electronic structure; EPR spectroscopy; Hydrogen bond; Thioether phenoxyl radical 1. Introduction Tyrosyl radicals play a vital role in the function of many biological systems [1,2]. Galactose oxidase (GO) [3–10] is a post-translationally modified enzyme, the active site of which involves a mononuclear Cu centre ligated by two his- tidines, two tyrosines, and one exogenous ligand (solvent or small molecule) in distorted square pyramidal coordination. The X-ray crystallographic studies have revealed an unusual post-translational modification comprising a cross-link between one ortho-position of Tyr 272 and Cys 228 [3,4]. This cross-link forms spontaneously in the presence of Cu(I) and O 2 [11,12], facilitates the oxidation of Tyr 272 [3,4] and is essential for the activity of GO [9]. Å Tyr 272 in Cu-free, oxi- dized, apo-GO [7–9,13] manifests an unusual EPR spectrum with a signal that is essentially axial (g 1 ¼ 2:0074; g 2 ¼ 2:0064; g 3 ¼ 2:0021) rather than rhombic, as is generally 0162-0134/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2007.07.013 * Corresponding authors. Tel.: +44 0 115 951 4188; fax: +44 0 115 951 3563 (C.D. Garner); Tel.: +44 0 115 951 3498; fax: +44 0 115 951 3563 (J. McMaster). E-mail addresses: benisvy@yahoo.com (L. Benisvy), Dave.Garner@ nottingham.ac.uk (C.D. Garner), j.mcmaster@nottingham.ac.uk (J. McMaster), neese@thch.uni-bonn.de (F. Neese). www.elsevier.com/locate/jinorgbio Available online at www.sciencedirect.com Journal of Inorganic Biochemistry 101 (2007) 1859–1864 JOURNAL OF Inorganic Biochemistry