We propose to investigate the 3 dimensional structure, active site architecture, catalytic mechanism, and mechanism of regulation of the soluble form of Methane Monooxygenase (MMO). This enzyme catalyzes the definitive first step in the oxidation of CH4 to CO2 by methanotrophic bacteria. In this way, the atmospheric egress of nearly all of the enormous quantity of CH4 (a potent """"""""greenhouse"""""""" gas) generated by anaerobic bacteria in aquatic environments is prevented. MMO also adventitiously catalyzes the oxidation of many other saturated and unsaturated hydrocarbons. Although the detailed mechanism of MMO is unknown, our studies suggest that the reaction is catalyzed by a cofactor not found in other oxygenases; this implies a new strategy for oxygenase catalysis. We have purified MMO from the type II methanotroph, Methylosinus trichosporium OB3b; it is composed of 3 proteins termed hydroxylase, reductase, and component B. The system offers many advantages over other purified MMO systems including greater yield and stability, and a 25-fold increase in hydroxylase specific activity. These properties allow purification in quantity so that biophysical techniques (optical EPR, Mossbauer, EXAFS, ENDOR, MCD, and CD spectroscopies) can be applied for structural studies. Recently, satisfactory crystals for structural studies have been obtained. Spectroscopy of small ligand complexes, isotopically labeled substrates and inhibitors, and transient kinetics are being used to investigate the molecular mechanism. Coordinated spectroscopic, chemical, and single turnover studies, have shown that the reaction is catalyzed by a mu-(R- or H-)oxo-bridged dinuclear Fe center located in the hydroxylase. We hypothesize that O2 adds to the [Fe(II)-Fe(II)] state of this cluster resulting in heterolytic O-O bond cleavage to form a reactive intermediate, perhaps an [Fe(IV)-Fe(IV)=O] oxene. This species is thought to attack hydrocarbons with the intermediate formation of a substrate radical. Substantial support for this mechanism is being accumulated through the use of specially synthesized chiral substrates for the detection of substrate radicals, the elucidation of characteristic peroxide shunt chemistry, and the detection of transient reaction intermediates. Catalytically active subsystems of MMO consisting of the hydroxylase without one or both of the other two components are being used to evaluate the mechanistic roles of the reductase and component B. Preliminary results suggest that these components play roles in both transfer of reducing equivalents necessary for catalysis, and in assuring efficient coupling of energy expenditure with methane turnover. This work should yield a fundamental understanding of a new type of biological oxygen activation chemistry, a new role for iron in this chemistry, and guidance in the design of catalyst for oxidation of abundant hydrocarbons. It is also likely to contribute to our understanding of the role of protein-protein interactions in biological regulatory processes. Moreover, as we have shown that priority pollutants such as trichloroethylene are rapidly oxidized and detoxified by MMO, it is probable that the knowledge gained from this study will find environmental applications.
| Komor, Anna J; Jasniewski, Andrew J; Que, Lawrence et al. (2018) Diiron monooxygenases in natural product biosynthesis. Nat Prod Rep 35:646-659 |
| Oloo, Williamson N; Banerjee, Rahul; Lipscomb, John D et al. (2017) Equilibrating (L)FeIII-OOAc and (L)FeV(O) Species in Hydrocarbon Oxidations by Bio-Inspired Nonheme Iron Catalysts Using H2O2 and AcOH. J Am Chem Soc 139:17313-17326 |
| Castillo, Rebeca G; Banerjee, Rahul; Allpress, Caleb J et al. (2017) High-Energy-Resolution Fluorescence-Detected X-ray Absorption of the Q Intermediate of Soluble Methane Monooxygenase. J Am Chem Soc 139:18024-18033 |
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| Lipscomb, John D (2014) Life in a sea of oxygen. J Biol Chem 289:15141-53 |
| Makris, Thomas M; Knoot, Cory J; Wilmot, Carrie M et al. (2013) Structure of a dinuclear iron cluster-containing ?-hydroxylase active in antibiotic biosynthesis. Biochemistry 52:6662-71 |
| Banerjee, Rahul; Meier, Katlyn K; Munck, Eckard et al. (2013) Intermediate P* from soluble methane monooxygenase contains a diferrous cluster. Biochemistry 52:4331-42 |
| Vu, Van V; Makris, Thomas M; Lipscomb, John D et al. (2011) Active-site structure of a ýý-hydroxylase in antibiotic biosynthesis. J Am Chem Soc 133:6938-41 |
| Makris, Thomas M; Chakrabarti, Mrinmoy; Münck, Eckard et al. (2010) A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis. Proc Natl Acad Sci U S A 107:15391-6 |
| Mitic, Natasa; Schwartz, Jennifer K; Brazeau, Brian J et al. (2008) CD and MCD studies of the effects of component B variant binding on the biferrous active site of methane monooxygenase. Biochemistry 47:8386-97 |
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