Structure and peroxidase activity of ferric streptomyces clavuligerus orf10-encoded protein P450CLA: UV-Visible, CD, MCD and EPR spectroscopic characterization

Abstract

The present study reports the spectroscopic characterization by UV-visible absorption spectroscopy, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) of the recombinant orf10-encoded P450-camphor like protein (P450CLA) of Streptomyces clavuligerus expressed in Escherichia coli Rosetta in the native form and associated to external ligands containing the ß-lactam, oxazole and alkylamine-derived (alcohol) moieties of the clavulamic acid. Considering the diversity of potential applications for the enzyme, the reactivity with tert-butylhydroperoxide (tert-BuOOH) was also characterized. P450CLA presents a covalently bound heme group and exhibited the UV-visible, CD and MCD spectral features of P450CAM including the fingerprint Soret band at 450 nm generated by the ferrous CO-complex. P450CLA was converted to high valence species by tert-BuOOH and promoted homolytic scission of the O-O bond. The radical profile of the reaction was tert-butyloxyl as primary and methyl and butylperoxyl as secondary radicals. The secondary methyl and butylperoxyl radicals resulted respectively from the ß-scission of the alkoxyl radical and from the reaction of methyl radical with molecular oxygen. © 2012 Sociedade Brasileira de Química.