Kinetic and spectroscopic characterization of intermediates in the soluble methane monooxygenase catalytic cycle: the old and the new

Abstract

Soluble methane monooxygenase (sMMO) catalyzes the difficult reaction of oxidative hydroxylation of methane to methanol, thereby allowing the host methanotroph to grow on methane as the sole source of carbon and energy. As methane possesses the strongest aliphatic C-H bond, the chemical mechanism of this enzyme is the epitome of oxygen activation in metalloenzymes. Exhaustive research of the catalytic mechanism of sMMO in recent years has elucidated many aspects of the chemistry occurring at the dinuclear iron active site center. These studies include the three dimensional structures of the protein components of sMMO and fairly detailed outlines of both the chemical mechanism and the method of regulatory control. The discovery of a diferric peroxo intermediate P, and more crucially, a high valent bis-μ-oxo diiron(IV) intermediate Q have provided the greatest advances in the understanding of oxygen activation in diiron oxygenases. The research described here has extended this understanding in several aspects. A long standing issue of low accumulation of enzyme reaction intermediates for spectroscopic studies has been addressed and in part solved. A new intermediate P* that had been proposed to exist based on kinetic studies has been trapped and characterized through spectroscopic techniques. P* appears to be a diferrous cluster intermediate that binds O2 weakly with little transfer of charge density onto the oxygen atoms. This result suggests a general theme for heme and non-heme oxygen activating enzymes in which ferrous centers initially form a weak complex with O2, which is strengthened in following steps by interactions such as stabilizing hydrogen bonds and charge donation from trans ligands. The long sought after goal of characterization of the vibrational spectrum of compound Q has been achieved through a time resolved resonance Raman technique. The preliminary results corroborate the diamond core structure that has been proposed for Q. Another putative high valent intermediate Q' has been discovered to arise from compound Q in the catalytic cycle. This is potentially a very significant finding as it seem likely that Q' rather than Q reacts directly with substrate. Following from a precedent from synthetic diiron model compound studies, it is possible that Q' is an open core form of the Q intermediate in which a reactive, terminal Fe(IV)=O moiety replaces the more stable bis-μ-oxo bridging structure.