Electron Paramagnetic Resonance Characterization of Three Iron–Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB from <i>Methanocaldococcus infernus</i> WilcoxenJarett ArragainSimon ScandurraAlessandro A. Jimenez-VicenteEmilio Echavarri-ErasunCarlos PollmannStephan BrittR. David RubioLuis M. 2016 NifB utilizes two equivalents of <i>S</i>-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe–S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron–molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen <i>Methanocaldococcus infernus</i> is a radical SAM enzyme able to reductively cleave SAM to 5′-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe–4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation. Nitrogen coordination to one or more of the auxiliary clusters in NifB was observed, and its mechanistic implications for NifB-co dissociation from the maturase are discussed.