The electron paramagnetic resonance characterisation of a copper-containing extracellular peroxidase from Thermomonospora fusca BD25

The actinomycete Thermomonospora fusca BD25 contains a peroxidase with a hi gh activity over a broad range of temperature and pH and a high stability a gainst denaturing agents. Unusually this peroxidase (PO) is a non-haem enzy me. 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 t he purified enzyme was assayed using H2O2 and 2,4-dichlorophenol (DCP). The intensity of the free radical EPR signal correlated with the peroxidase ac tivity in a variety of enzyme preparations. Furthermore, when DCP and H2O2 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 t he broad signal with the oxidation of the preparation since a similar incre ase can be achieved by the addition of ferricyanide. The high intensity of the broad signal in the ferricyanide treated PO allowed us to deconvolute t he signal into several components using the difference in their relaxation characteristics: two distinct copper signals were detected, one of which wa s similar to a type 2 centre. Furthermore a symmetrical singlet was detecte d at g = 2.059, consistent with the presence of an iron complex with a high degree of symmetry and weakly coordinated ligands. (C) 1999 Elsevier Scien ce B.V. All rights reserved.