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.
The metalloenzymes we study raise profound, fundamental issues regarding the way in which metal centers transform their substrates, while playing a major role in the world’s food supply (nitrogenase) and in disease states (biosynthesis of molybdoenzymes). The agronomic, economic, and social significance of the enzyme nitrogenase can be appreciated by recognizing that the lives of about two-thirds of today’s human population depend on plant growth which relies on biologically fixed nitrogen generated by nitrogenase. The other third of the population depends on nitrogen fixation by the industrial Haber-Bosch process, but this process is a significant component in the world’s energy usage, demanding approximately 1% of human energy consumption. Genetic deficiencies in human biosynthesis of the molybdenum cofactor (Moco) lead to neurological damage that commonly results in death, and most of these come from mutations to the radical-SAM enzyme MOCS1A. We study MOCS1A and its bacterial equivalent MoaA (more conveniently accessible), which participate in the first step in the synthesis of Moco. Our unique capacity to perform and analyze advanced paramagnetic resonance spectroscopic measurements (ENDOR, ESEEM) provides a powerful, selective, and sensitive tool for the characterization of the active sites of such metalloenzymes, and their interactions with substrates/inhibitors/products. By examining both the resting state and key catalytic intermediates our studies make key contributions to the determination of the mechanism of function of these enzymes. This laboratory continues to pioneer in the development of ENDOR, ESEEM as applied to metal ions in biological systems. It acts as a resource to the community in part through formal and informal collaborations, and in the training of the next generation of practitioners as undergraduates, graduate students and postdoctorals. A part of this program has been a strenuous effort to incorporate underrepresented groups, with considerable success having been achieved in attaining gender and geographic diversity, and limited success in achieving ethnic diversity.
