Nitrogenases are vitally important enzymes that perform an amazing chemical reaction, the reduction of N2 to ammonia. In nitrogenases, iron-sulfur clusters catalyze multielectron reductions of small molecules, a role that differs from the more common role of iron-sulfur clusters as electron-transfer sites that avoid bond-making and bond-breaking reactions. Little is known about the mechanism of substrate reduction by nitrogenases. Our guiding hypothesis is that nitrogenases and other reactive iron-sulfur cluster enzymes generate a transient open site on an iron atom for substrate binding. Recent spectroscopic work on iron- molybdenum nitrogenase strongly suggests that the mechanism involves binding of N2 to iron, and involves iron-hydride species. However, there are no chemical precedents for iron-sulfur clusters that have an open site, or for iron-sulfur clusters with a hydride. Examples of N2 binding to high-spin iron are rare and understudied. Synthetic compounds with these features are needed to evaluate the feasibility of the proposed functional groups on iron-sulfur clusters, to establish the spectroscopic signatures of these functional groups, and to learn whether their reactivity is consistent with the enzymatic products. In the proposed research, we will create synthetic iron-containing compounds with each of these functionalities: unsaturated iron-sulfur clusters, iron-sulfide-hydride clusters, and iron-N2 complexes. The isolation and characterization of these compounds is made possible by the use of very bulky supporting groups. The bulky groups also facilitate crystallization, and enhance solubility in organic solvents that can be used at low temperature. Crystallography, kinetic studies, electrochemistry, and reactivity will be used to elucidate the atomic-level detail of the elementary steps of small-molecule binding and reduction. The synthetic complexes will be evaluated by ENDOR, infrared, Raman, M""""""""ssbauer, and X- ray absorption spectroscopies to provide a link between the structures of novel model compounds and the known data for nitrogenases. We anticipate that the proposed work will lead to the first solid precedents for reaction pathways in nitrogenases. Although much is known about the mechanisms of multielectron oxidation reactions in bioinorganic chemistry, the knowledge about multielectron biological reductions is much less. Therefore, there is fundamental importance in learning how the iron-sulfide cluster in nitrogenase binds and transforms small molecules that are essential for life. In the long run, understanding the mechanisms of small-molecule reduction in biological systems may also lead to new catalysts for use in chemical synthesis.

Public Health Relevance

Enzymes that contain iron and sulfur produce many of the molecules that are essential for life, but are not understood well. The iron-sulfur enzyme nitrogenase converts unreactive nitrogen in the atmosphere into forms that can be used, and life as we know it is dependent upon this process of nitrogen fixation. However, the scientific community does not yet know how nitrogenase works. This project aims to show the principles underlying the mechanism of nitrogen fixation, which may also lead to new catalysts for transforming organic and inorganic molecules.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM065313-09
Application #
8465238
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2004-04-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
9
Fiscal Year
2013
Total Cost
$303,674
Indirect Cost
$112,604
Name
Yale University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Broere, Daniel L J; Mercado, Brandon Q; Bill, Eckhard et al. (2018) Alkali Cation Effects on Redox-Active Formazanate Ligands in Iron Chemistry. Inorg Chem 57:9580-9591
Broere, Daniël L J; Holland, Patrick L (2018) Boron compounds tackle dinitrogen. Science 359:871
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DeRosha, Daniel E; Holland, Patrick L (2018) Incorporating light atoms into synthetic analogues of FeMoco. Proc Natl Acad Sci U S A 115:5054-5056
Broere, Daniël L J; Mercado, Brandon Q; Lukens, James T et al. (2018) Reversible Ligand-Centered Reduction in Low-Coordinate Iron Formazanate Complexes. Chemistry 24:9417-9425

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