Electron Paramagnetic Resonance Characterization of Three Iron-Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB from Methanocaldococcus infernus.
NifB utilizes two equivalents of S-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 Methanocaldococcus infernus 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.
Department of Chemistry, University of California , Davis, California 95616, United States.
Jarett
J
Wilcoxen
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid , Pozuelo de Alarcón, Madrid 28223, Spain.
Simon
S
Arragain
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid , Pozuelo de Alarcón, Madrid 28223, Spain.
Alessandro A
AA
Scandurra
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid , Pozuelo de Alarcón, Madrid 28223, Spain.
Emilio
E
Jimenez-Vicente
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid , Pozuelo de Alarcón, Madrid 28223, Spain.
Carlos
C
Echavarri-Erasun
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid , Pozuelo de Alarcón, Madrid 28223, Spain.
Stephan
S
Pollmann
Department of Chemistry, University of California , Davis, California 95616, United States.
R David
RD
Britt
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid , Pozuelo de Alarcón, Madrid 28223, Spain.
Luis M
LM
Rubio
D001426:Bacterial Proteins / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D058085:Iron Compounds / Q000737:chemistry*; D063747:Methanocaldococcus / Q000201:enzymology*; D008983:Molybdoferredoxin / Q000737:chemistry; D009591:Nitrogenase / Q000737:chemistry*; D012436:S-Adenosylmethionine / Q000737:chemistry*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D058085:Iron Compounds; D008983:Molybdoferredoxin; C085381:NifB cofactor; C092498:NifB protein, Bacteria; D012436:S-Adenosylmethionine; D009591:Nitrogenase