Biophysical Studies of Metalloenzymes

Northwestern University, Evanston IL

Investigators

Abstract

Biological nitrogen fixation - the reduction of N2 to yield ammonia - is catalyzed by a complex metalloenzyme called nitrogenase. The agronomic, economic, and social significance of this enzyme can be appreciated by recognizing that the lives of about two-thirds of today''s human population depend on plant growth that involves biologically fixed nitrogen. Nitrogenase is, however, the most complex metalloenzyme, and despite nearly 40 years of intense investigation, it has repulsed all efforts to characterize the mechanism of biological nitrogen reduction, and nothing was known about the intermediate stages in the process. Recently, however, this project has participated in the first successes at trapping catalytic intermediates, and it has obtained the first details of their structure. This information is acquired through the use of electron-nuclear double resonance (ENDOR) and electron spinecho envelope modulation (ESEEM) spectroscopies. These techniques permit the NMR determination of the complete coordination geometry and bonding of the metal ions of the N2-binding/reduction active site, the FeMo-cofactor ([7Fe-9S-Mo-X-homocitrate], and can supply detailed structures of metal-bound substrates, intermediates, and products. This project has three components: (i) ENDOR/ESEEM Characterization of Trapped N2 Reduction Intermediates: This research aims to determine the structures of substrate-derived species bound to the active-site FeMo-cofactor in multiple intermediates throughout the catalytic cycle of nitrogenase. (ii) Electronic Structure of the Active-Site Molybdenum-Iron cofactor (FeMo-co) Active Site. The parallel goal is to determine the metal-ion valencies of FeMo-co throughout the catalytic cycle. (iii)The Identity of the Interstitial Atom of FeMo-co. Long after the basic structure of FeMo-co had been determined, a higher-resolution X-ray structure revealed the presence of a single N, O, or C atom at its center. This component of the project is aimed at determining the identity of this atom. Broader Impact: This project will involve pioneering development of the tools, ENDOR and ESEEM, and of the analysis procedures and algorithms that together give these techniques their power. The development of these capabilities is diseminated through publications and presentations, through formal and informal collaborations, as well as distribution of analysis programs via the web. Most importantly, it is involved in training the next generation of practitioners as graduate students and postdoctoral fellows. Through these contributions it is fundamentally altering the disciplines of metallobiochemistry/bioinorganic chemistry.