Biophysical Studies of Metalloenzymes

Northwestern University, Evanston IL

Investigators

Abstract

Electron paramagnetic resonance spectroscopy has long been a central tool for determining the structure and function of metalloenzyme active sites. This project will continue to develop unique methods to perform CW/pulsed ENDOR and ESEEM experiments at X (9 GHz) and Q (35 GHz) bands, and W band (95 GHz) microwave frequencies. This constellation of approaches, combined with new procedures developed for the analysis of frozen-solution ENDOR and ESEEM spectra, provides a powerful, selective, and sensitive tool for the characterization of metalloenzyme active sites and their interactions with substrates/inhibitors/products, both in the resting state and in key intermediates. This project will emphasize three important topics: (I) Localization of substrate/inhibitor binding at the molybdenum-iron cofactor of the nitrogenase MoFe protein and characterization of intermediate states formed during substrate reduction; (II) The enzymic mechanism of three members of "radical S-adenosylmethionine" superfamily, which all use [4Fe-4S] clusters and S-adenosylmethionine (AdoMet) to generate catalytically essential radicals. (III) Reactive intermediates and substrate/inhibitor interactions with molybdenum and tungsten oxotransferases (molybdopterins). Broader Impact: This project will develop sophisticated analytical procedures and algorithms for determining the complete coordination geometry and bonding at a metal center. These capabilities disseminated to the metallobiochemistry researchers via web, through formal collaborations and by informal consultation. Besides these research contributions, the project will provide research opportunities for a faculty member from an undergraduate institution. In addition, the project will involve training at the graduate and postdoctoral level. This project is funded jointly by Molecular Biochemistry and Molecular Biophysics.