Methane Monooxygenase

Abstract

The X-ray crystal structure determination of the resting state of the hydroxylase enzyme of methane monooxygenase from the organism Methylococcus capsulatus (Bath) reveals a dinuclear iron center bridged by a hydroxide ion, an acetate ligand from the crystallization buffer, and the carboxylate group of a glutamate residue. The terminal positions of this diiron(III) unit are occupied by three additional glutamates, two histidines, and a water molecule. Residues adjacent to this bioinorganic active site form a hydrophobic pocket for methane binding. The reaction cycle of the enzyme begins with reduction to the diiron(II) state in the presence of substrate and two other protein components, a reductase and a small molecular weight coupling protein, also known as protein B. Reaction of the reduced hydroxylase with dioxygen proceeds through several intermediates prior to hydroxylation of alkane to yield the product alcohol, water, and the resting diiron(III) form of the enzyme. The rate of the reaction, regioselectivity of hydroxylation, and ability to detect the intermediates spectroscopically depends upon the presence of the coupling protein. Freeze-quench Mössbauer and low temperature stopped-flow optical spectroscopic experiments have identified two key intermediates in the reaction cycle, designated L and Q, the properties of which are discussed. Experiments with radical clock substrate probes revealed no evidence for substrate radical intermediates. Mechanisms are proposed to account for these observations.