XAS STUDIES OF ACTIVE SITES AND COMPONENT INTERACTIONS IN BACTERIAL MULTICOMPONE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to probe systematically the electronic and geometric structure of the carboxylate-bridged nonheme diiron sites in a series of bacterial multicomponent monooxygenases (BMMs) using x-ray absorption spectroscopy (XAS). BMMs comprise a family of enzymes capable of utilizing molecular oxygen to oxidize a variety of hydrocarbon substrates, and require complexes between several protein components for efficient catalysis. Our goal is to elucidate the role of component interactions and structural flexibility at the diiron active center in the catalytic function and substrate specificity of BMMs. The proteins systems studied will include soluble methane monooxygenase (sMMO) from M. capsulatus (Bath), toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. Our previous XAS studies of the sMMO and ToMO enzymes have afforded a structural characterization of the diiron site in the hydroxylase component, and identified its changes upon interaction with the regulatory proteins. We plan to extend XAS experiments to explore other protein-protein interactions in these systems and in the novel phenol hydroxylase. Specifically, we propose i) to obtain electronic and structural information on the diiron active center in intermediate species formed in the reaction of the hydroxylase component of BMMs with dioxygen; ii) to analyze the electronic and geometric structure of the dinuclear iron site in different oxidation states in the three hydroxylases; iii) to elucidate the effect of the coupling protein on the active center in the phenol hydroxylase; and iv) to determine how the binding between the hydroxylase and reductase component containing the [2Fe-2S] cluster affects the electronic and metrical characteristics of the dimetal active site and/or [2Fe-2S] center in the three multicomponent monooxygenases.