ABDUCTION OF IRON(III) FROM THE SOLUBLE METHANE MONOOXYGENASE HYDROXYLASE AND RECONSTITUTION OF THE BINUCLEAR SITE WITH IRON AND MANGANESE

The apo-form of the soluble methane monooxygenase hydroxylase from Met hylococcus capsulatus (Bath) was prepared via chelation of iron(III) w ith 3,4-dihydroxybenzaldehyde. The apohydroxylase was reconstituted by the anaerobic addition of Fe(II) followed by air oxidation. The enzym e thus prepared regained 85-90% of its original catalytic activity. Th e incorporation of two manganese(II) ions/mol of apohydroxylase was mo nitored 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 mu-h ydroxo (alkoxo) bridge. The results of this study indicate that the M. capsulatus (Bath) hydroxylase contains a single diiron site.