Complex Formation between the Protein Components of Methane Monooxygenase from Methylosinus trichosporium OB 3 b

Kinetic, spectroscopic, and chemical evidence for the formation of specific catalytically essential complexes between the three protein components of the soluble form of methane monooxygenase from Methylosinus trichosporiurn OB3b is reported. The effects of the concentrations of the reductase and component B on the hydroxylation activity of the reconstituted enzyme system has been numerically simulated based on a kinetic model which assumes formation of multiple high affinity complexes with the hydroxylase component during catalysis. The formation of several of these complexes has been directly demonstrated. By using EPR spectroscopy, the binding of approximately 2 mol of component B/mol of hydroxylase (subunit structure (cY&)z) is shown to significantly change the electronic environment of the p-(H/R)-oxo-bridged binuclear iron cluster of the hydroxylase in both the mixed valent (Fe(II)*Fe(III)) and fully reduced (Fe(II)*Fe(II)) states. Protein-protein complexes between the reductase and component B as well as between the reductase and hydroxylase have been shown to form by monitoring quenching of the tryptophan fluorescence spectrum of either the component B (KO = 0.4 PM) or hydroxylase (two binding sites, Koa = 10 nM, K D ~ = 8 p ~ ) . The observed K D values are in agreement with the best fit values from the kinetic simulation. Through the use of the covalent zero length cross-linking reagent l-ethyl3-(3-dimethylaminopropyl)-carbodiimide (EDC), the binding sites of the component B and reductase were shown to be on the hydroxylase CY and f i subunits, respectively. The CY and 0 subunits of the hydroxylase are cross-linked by EDC suggesting that they are juxtaposed. EDC also caused the rapid loss of the ability of the monomeric component B to stimulate the hydroxylation reaction suggesting that cross-linking of reactive groups on the protein surface had occurred. This effect was inhibited by the presence of hydroxylase and was accompanied by a loss of the ability of the component B to bind to the hydroxylase. Thus, formation of a component B-hydroxylase complex is appar-