In nitrogenases, iron-sulfur (FeS) clusters transcend their usual role as electron transfer sites, by performing the difficult multielectron reduction of N2 to NH3. Nitrogenase thus demonstrates the amazing catalytic ability of iron-sulfur clusters in biological systems. The site of N2 reduction in nitrogenase is an FeS cluster called the iron-molybdenum cofactor (FeMoco), which is the only example of a metal-carbide in biological chemistry. This carbide may be inserted by way of cluster-CH3, -CH2, and -CH intermediates, which are also unprecedented in FeS cluster chemistry. This enzyme is currently postulated to use species unknown to chemists: organometallic FeS clusters, FeS clusters with carbides, FeS clusters with hydrides, and FeS clusters bound to N2. Because of the lack of precedents, there is an urgent need to build up the experimental basis for evaluating the literature-proposed mechanisms for FeMoco biosynthesis and activity. Our guiding hypothesis is that the role of carbide in FeMoco is to hold and release transient low-coordinate iron sites, which can form bridging Fe-N2 and Fe-H intermediates during the catalytic mechanism. This will be tested using the synthetic analogue strategy, which is well-precedented in bioinorganic chemistry. Synthetic FeS clusters with N2, H, and C groups on the cluster can show the feasibility of the proposed functional groups on iron-sulfur clusters, establish the spectroscopic signatures of specific functional groups, and show whether their reactions are consistent with the models for FeMoco mechanism. Similarly, cluster-bound CH2, CH, and C groups would help to determine the feasibility of potential steps in FeMoco biosynthesis. In the proposed research, we will synthesize and study synthetic FeS compounds with each of the following novel functionalities: (1) unsaturated iron-sulfur clusters that bind nitrogenase substrates, (2) iron-sulfide- hydride clusters, and (3) iron clusters with CH2/CH/C bridges. We will use bulky supporting groups to stabilize reactive species, to facilitate crystallization, and to enable systematic study of their reactions. Crystallography, kinetic studies, electrochemistry, and reactivity will be used to understand the binding and reduction of N2 and other nitrogenase substrates. This in turn shows what types of reactions are reasonable to expect with the FeMoco. The structurally-defined synthetic complexes will also be evaluated by magnetic resonance, infrared, Raman, Mssbauer, and X-ray absorption spectroscopies, which will serve to translate the known spectroscopic data for nitrogenase into reasonable structural conclusions. Nitrogenase is one of the strangest metalloenzymes, because of its strongly reducing multielectron reduction, the cofactor structure with a carbide, and the ability to interact with usually-inert N2. Understanding its mechanism thus requires new discoveries about the fundamental chemistry of FeS clusters. This project aims to provide the chemical precedents that are needed to put nitrogenase mechanism on a firm footing.
The iron-sulfur enzyme nitrogenase 'fixes' nitrogen in the air by converting it into ammonia, and all life on Earth is dependent upon nitrogen fixation. We will make new cluster-containing molecules that reproduce attributes of the nitrogenase iron-sulfur active site, and these will teach us about the fundamental reactions of iron-sulfur clusters that enable the varied roles iron-sulfur clusters play in biochemical reactions.
| Broere, Daniël L J; Holland, Patrick L (2018) Boron compounds tackle dinitrogen. Science 359:871 |
| Skubi, Kazimer L; Holland, Patrick L (2018) So Close, yet Sulfur Away: Opening the Nitrogenase Cofactor Structure Creates a Binding Site. Biochemistry 57:3540-3541 |
| 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 |
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| Broere, Daniël L J; Mercado, Brandon Q; Holland, Patrick L (2018) Selective Conversion of CO2 into Isocyanate by Low-Coordinate Iron Complexes. Angew Chem Int Ed Engl 57:6507-6511 |
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| Pelmenschikov, Vladimir; Gee, Leland B; Wang, Hongxin et al. (2018) High-Frequency Fe-H Vibrations in a Bridging Hydride Complex Characterized by NRVS and DFT. Angew Chem Int Ed Engl 57:9367-9371 |
| McWilliams, Sean F; Bunting, Philip C; Kathiresan, Venkatesan et al. (2018) Isolation and characterization of a high-spin mixed-valent iron dinitrogen complex. Chem Commun (Camb) 54:13339-13342 |
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