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Journal of the Brazilian Chemical Society

versión impresa ISSN 0103-5053

Resumen

GOTO, Leandro S. et al. Structure and peroxidase activity of ferric Streptomyces clavuligerus orf10-encoded protein P450CLA: UV-visible, CD, MCD and EPR spectroscopic characterization. J. Braz. Chem. Soc. [online]. 2012, vol.23, n.5, pp. 913-920. ISSN 0103-5053.  http://dx.doi.org/10.1590/S0103-50532012000500017.

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 β-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 β-scission of the alkoxyl radical and from the reaction of methyl radical with molecular oxygen.

Palabras llave : cytochrome P450; orf10; peroxidase activity; magnetic circular dichroism; electron paramagnetic resonance; spin trapping.

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