The electron paramagnetic resonance characterisation of a copper- containing extracellular peroxidase from Thermomonospora fusca BD25 Dimitri A. Svistunenko, Abdul Rob, Andrew Ball, Jaume Torres, Martyn C.R. Symons, Michael T. Wilson, Chris E. Cooper * Department of Biological Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK Received 29 September 1998; received in revised form 7 July 1999; accepted 15 July 1999 Abstract The actinomycete Thermomonospora fusca BD25 contains a peroxidase with a high activity over a broad range of temperature and pH and a high stability against denaturing agents. Unusually this peroxidase (PO) is a non-haem enzyme. As prepared PO is characterised by two electron paramagnetic resonance (EPR) signals, detected at liquid helium temperature, a free radical signal (g = 2.0045) and a broad signal at g = 2.056. The peroxidase activity of the purified enzyme was assayed using H 2 O 2 and 2,4-dichlorophenol (DCP). The intensity of the free radical EPR signal correlated with the peroxidase activity in a variety of enzyme preparations. Furthermore, when DCP and H 2 O 2 were added to PO a significant increase of both the free radical signal and the broad signal at g = 2.056 was observed. We associate the increase of the broad signal with the oxidation of the preparation since a similar increase can be achieved by the addition of ferricyanide. The high intensity of the broad signal in the ferricyanide treated PO allowed us to deconvolute the signal into several components using the difference in their relaxation characteristics : two distinct copper signals were detected, one of which was similar to a type 2 centre. Furthermore a symmetrical singlet was detected at g = 2.059, consistent with the presence of an iron complex with a high degree of symmetry and weakly coordinated ligands. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : Non-heme ; Peroxidase ; Thermomonospora fusca BD25; Electron paramagnetic resonance; Copper; Iron; Free radical 1. Introduction Peroxidases catalyse the two electron oxidation of a variety of substrates in the presence of hydrogen peroxide. Haem is considered a traditional motif of peroxidases, as it is the site where the primary event of electron transfer to H 2 O 2 takes place. However, a number of intracellular enzymes with peroxidase ac- tivity have been found to contain no haem, for ex- ample intracellular peroxidases from Ascophilum nodosum [1], Streptomyces aureofaciens [2], Pseudo- monas pyrrocina [3], Corallina o¤cinalis [4] and Ser- ratia mascescens [5]. Extracellular peroxidases have only been identi¢ed in a few genera of micro-organisms, including ligni- nolytic fungi. The most extensively studied lignino- lytic fungi, Phanerochaete chrysosporium secrete two 0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0167-4838(99)00163-6 Abbreviations : PO, Thermomonospora fusca peroxidase (as prepared, i.e. undialysed, if not indicated otherwise) ; DCP, (2,4-dichlorophenol) ; SDS, sodium dodecyl sulphate ; EPR, elec- tron paramagnetic resonance; IR, infrared; T, temperature of EPR measurement (K) ; X, microwave frequency (GHz); P, mi- crowave power (mW) ; A m , modulation amplitude (G); R, EPR spectra sweep rate (G/s); d, time constant (ms); NS, number of scans in EPR spectra acquisition; S, line shape symmetry param- eter * Corresponding author. Fax: +44-1206-872592; E-mail : ccooper@essex.ac.uk Biochimica et Biophysica Acta 1434 (1999) 74^85 www.elsevier.com/locate/bba