Reduced nitrogen is an essential component of nucleic acids and proteins. Therefore, all organisms require this nutrient for growth. Unfortunately, even though elemental dinitrogen (N2) comprises 79% of the earth's atmosphere, this abundant source is inert and can only be mobilized for biosynthesis following its conversion to a usable form like ammonia. In nature, this N2 fixation ability is restricted to a small but diverse group of diazotrophic microorganisms. Diazotrophs have in common the enzyme nitrogenase, which is the subject of this proposal, and which catalyses the MgATP-dependent reduction of N2 to ammonia. Nitrogenase is a complex metalloprotein composed of two separately purifiable protein components, the iron (Fe) protein and the molybdenum-iron (MoFe) protein, both of which containing metal cluster(s). Although it is well established that metalloproteins play a variety of essential roles in the catabolic and metabolic regulation as well as metal storage, very little is known about the biosynthesis of their metal centers and the mechanism through which these metal clusters are incorporated into proteins. Nitrogenase is no exception, being perhaps the most intriguing and complicated metalloprotein isolated so far. Another aspect of nitrogenase study that draws considerable attention involves its catalytic mechanism. How energy transduction, a fundamentally important process, is correlated with conformational changes of the protein, allowing it to carry out its catalytic function is not fully understood so far. Here we propose to greatly expand our understanding of the nitrogenase assembly and catalytic mechanism by combined genetic, biochemical and biophysical approaches. The organism of interest is Azotobacter vinelandii, one of the diazotrophs producing the molybdenum nitrogenase. The main focus of the proposed investigation will be the assembly process of nitrogenase MoFe protein, which contains two complex and unique metal clusters, P-cluster and FeMoco. Meanwhile, questions regarding the catalytic mechanism of nitrogenase will also be addressed in this study, with the nitrogenase Fe protein and its interaction with nucleotides as the center of attention. Our proposed studies will endeavor to greatly broaden our knowledge on the catalytic mechanism of Fe protein and improve our understanding on one of the fundamental issues in biology: energy transduction, which involves the switching of protein between conformational states upon nucleotide binding and its subsequent hydrolysis.
| Rettberg, Lee A; Wilcoxen, Jarett; Lee, Chi Chung et al. (2018) Probing the coordination and function of Fe4S4 modules in nitrogenase assembly protein NifB. Nat Commun 9:2824 |
| Tanifuji, Kazuki; Lee, Chi Chung; Sickerman, Nathaniel S et al. (2018) Tracing the 'ninth sulfur' of the nitrogenase cofactor via a semi-synthetic approach. Nat Chem 10:568-572 |
| Rupnik, Kresimir; Lee, Chi Chung; Hu, Yilin et al. (2018) A VTVH MCD and EPR Spectroscopic Study of the Maturation of the ""Second"" Nitrogenase P-Cluster. Inorg Chem 57:4719-4725 |
| Sickerman, Nathaniel S; Rettberg, Lee A; Lee, Chi Chung et al. (2017) Cluster assembly in nitrogenase. Essays Biochem 61:271-279 |
| Fay, Aaron W; Blank, Michael A; Rebelein, Johannes G et al. (2016) Assembly scaffold NifEN: A structural and functional homolog of the nitrogenase catalytic component. Proc Natl Acad Sci U S A 113:9504-8 |
| Hu, Yilin; Ribbe, Markus W (2016) Maturation of nitrogenase cofactor-the role of a class E radical SAM methyltransferase NifB. Curr Opin Chem Biol 31:188-94 |
| Lee, Chi Chung; Sickerman, Nathaniel S; Hu, Yilin et al. (2016) YedY: A Mononuclear Molybdenum Enzyme with a Redox-Active Ligand? Chembiochem 17:453-5 |
| Lee, Chi Chung; Fay, Aaron W; Weng, Tsu-Chien et al. (2015) Uncoupling binding of substrate CO from turnover by vanadium nitrogenase. Proc Natl Acad Sci U S A 112:13845-9 |
| Wiig, Jared A; Hu, Yilin; Ribbe, Markus W (2015) Refining the pathway of carbide insertion into the nitrogenase M-cluster. Nat Commun 6:8034 |
| Cahn, Jackson K B; Brinkmann-Chen, Sabine; Spatzal, Thomas et al. (2015) Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases. Biochem J 468:475-84 |
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