Probing the coordination and function of Fe 4 S 4 modules in nitrogenase assembly protein NifB

• Published in: Nature communications 2018-07, Vol.9 (1), p.2824
• Main Authors: Rettberg, Lee A, Wilcoxen, Jarett, Lee, Chi Chung, Stiebritz, Martin T, Tanifuji, Kazuki, Britt, R David, Hu, Yilin
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Binding Sites; Cloning, Molecular; Electron Spin Resonance Spectroscopy; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Iron - chemistry; Iron - metabolism; Iron Compounds - chemistry; Iron Compounds - metabolism; Methanosarcina - chemistry; Methanosarcina - metabolism; Models, Molecular; Nitrogenase - chemistry; Nitrogenase - genetics; Nitrogenase - metabolism; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; S-Adenosylmethionine - chemistry; S-Adenosylmethionine - metabolism; Sequence Alignment; Substrate Specificity; Sulfur - chemistry; Sulfur - metabolism
• ISSN: 2041-1723

NifB is an essential radical S-adenosylmethionine (SAM) enzyme for nitrogenase cofactor assembly. Previous studies show that NifB couples a putative pair of [Fe S ] modules (designated K1 and K2) into an [Fe S C] cofactor precursor concomitant with radical SAM-dependent carbide insertion through the action of its SAM-binding [Fe S ] module. However, the coordination and function of the NifB cluster modules remain unknown. Here, we use continuous wave and pulse electron paramagnetic resonance spectroscopy to show that K1- and K2-modules are 3-cysteine-coordinated [Fe S ] clusters, with a histidine-derived nitrogen serving as the fourth ligand to K1 that is lost upon K1/K2-coupling. Further, we demonstrate that coexistence of SAM/K2-modules is a prerequisite for methyltransfer to K2 and hydrogen abstraction from the K2-associated methyl by a 5'-deoxyadenosyl radical. These results establish an important framework for mechanistic explorations of NifB while highlighting the utility of a synthetic-cluster-based reconstitution approach employed herein in functional analyses of iron-sulfur (FeS) enzymes.