Abduction of iron(III) from the soluble methane monooxygenase hydroxylase and reconstitution of the binuclear site with iron and manganese

Abstract

The apo‐form of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath) was prepared via chelation of iron(III) with 3,4‐dihydroxybenzaldehyde. The apohydroxylase was reconstituted by the anaerobic addition of Fe(II) followed by air oxidation. The enzyme thus prepared regained 85–90% of its original catalytic activity. The incorporation of two manganese(II) ions/mol of apohydroxylase was monitored by EPR spectroscopy. The Mn(II) ions occupy the native diiron active site and remain in the +2 oxidation state. The EPR data suggest strong coupling between the two Mn(II) ions and retention of the μ‐hydroxo (alkoxo) bridge. The results of this study indicate that the M. capsulatus (Bath) hydroxylase contains a single diiron site. Copyright © 1993, Wiley Blackwell. All rights reserved