CRYSTALLOGRAPHIC ANALYSIS OF METHANE MONOOXYGENASE SYS

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This project is a collaborative effort between our laboratory and that of Stephen J. Lippard at the Massachusetts Institute of Technology. The major objective of the research is to elucidate the three dimensional structure of the proteins involved in the conversion of methane to methanol in methanotrophic bacterium, Methylococcus capsulatus (Bath). This soluble methane monooxygenase system (MMO) has three major components, a multi-subunit hydroxylase, a reductase, and an electron transfer coupling protein, protein B. The active hydroxylase is a dimer with each protomer composed of three polypeptide chains such that the entire enzyme has an (alpha beta gamma) 2 configuration and a total molecular weight of roughly 250kDa. We have determined the structure of the hydroxylase to 2.2 Angstroms resolution. One major focus of the current proposal is to extend the structure of this protein to the limit of resolution of the native crystals (1.8 Angstroms). In addition, we plan initially to utilize this structural information to investigate the mechanism of oxygen activation by several closely related structure determinations. These include characterization of the reduced form of the hydroxylase and the iron depleted apo form of the protein. As a complement to solution mechanistic work on the MMO hydroxylase protein, crystals containing bound hydrocarbon substrate molecules will be studied to elucidate the interaction of substrate with the dinuclear iron core. Due to the availability of an overexpression system and purification scheme for protein B, and the strong evidence of its role in the mediation of the overall activation reaction, we plan next to attempt co- crystallization and structure determination of this complex as well. These detailed structural studies will facilitate an understanding of interactions with the other MMO proteins, the reductase and the coupling protein B, and will enable us to learn how the hydroxylase protein environment modulates the reaction chemistry of the diiron oxo center. The structural and functional similarity between the MMO hydroxylase and the small subunit of the enzyme ribonucleotide reductase (RR) suggest that information about the former will help to sharpen insights about RR, itself a target of antiviral and antitumor pharmaceuticals because of its pivotal function in the biosynthesis of DNA.