Structural Insights into the Mechanism of the Radical SAM Carbide Synthase NifB, a Key Nitrogenase Cofactor Maturating Enzyme

• Published in: Journal of the American Chemical Society Vol. 142; no. 25; pp. 11006 - 11012
• Main Authors: Fajardo, Ana Sosa, Legrand, Pierre, Payá-Tormo, Lucı́a, Martin, Lydie, Pellicer Martı́nez, Maria Teresa, Echavarri-Erasun, Carlos, Vernède, Xavier, Rubio, Luis M, Nicolet, Yvain
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 24-06-2020
• Subjects: Archaeal Proteins - chemistry; Archaeal Proteins - metabolism; Binding Sites; Biochemistry, Molecular Biology; Crystallography, X-Ray; Cysteine - chemistry; Glutamic Acid - chemistry; Histidine - chemistry; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - metabolism; Life Sciences; Methanosarcinales - enzymology; Models, Chemical; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Protein Binding; Protein Conformation; S-Adenosylmethionine - metabolism; Structural Biology
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.0c02243

Nitrogenase is a key player in the global nitrogen cycle, as it catalyzes the reduction of dinitrogen into ammonia. The active site of the nitrogenase MoFe protein corresponds to a [MoFe7S9C-(R)-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF (for NItrogen Fixation) assembly machinery. Among them, the radical SAM protein NifB plays an essential role, concomitantly inserting a carbide ion and coupling two [Fe4S4] clusters to form a [Fe8S9C] precursor called NifB-co. Here we report on the X-ray structure of NifB from Methanotrix thermoacetophila at 1.95 Å resolution in a state pending the binding of one [Fe4S4] cluster substrate. The overall NifB architecture indicates that this enzyme has a single SAM binding site, which at this stage is occupied by cysteine residue 62. The structure reveals a unique ligand binding mode for the K1-cluster involving cysteine residues 29 and 128 in addition to histidine 42 and glutamate 65. The latter, together with cysteine 62, belongs to a loop inserted in the active site, likely protecting the already present [Fe4S4] clusters. These two residues regulate the sequence of events, controlling SAM dual reactivity and preventing unwanted radical-based chemistry before the K2 [Fe4S4] cluster substrate is loaded into the protein. The location of the K1-cluster, too far away from the SAM binding site, supports a mechanism in which the K2-cluster is the site of methylation.