title: In vitro synthesis of the iron-molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins.
abstract: Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron-molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 9S, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from Fe(2+), S(2-), MoO4(2-), and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with Fe(2+), S(2-), MoO4(2-), R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed.
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Curatti
authors.forename: Jose A
authors.initials: JA
authors.lastname: Hernandez
authors.forename: Robert Y
authors.initials: RY
authors.lastname: Igarashi
authors.forename: Basem
authors.initials: B
authors.lastname: Soboh
authors.forename: Dehua
authors.initials: D
authors.lastname: Zhao
authors.forename: Luis M
authors.initials: LM
authors.lastname: Rubio
mesh_terms: D016948:Azotobacter vinelandii / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D007202:Indicators and Reagents; D007501:Iron; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D008982:Molybdenum; D008983:Molybdoferredoxin / Q000138:chemical synthesis*; D009586:Nitrogen Fixation; D009591:Nitrogenase / Q000378:metabolism*; D013455:Sulfur; D014233:Tricarboxylic Acids
chemical_list: D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008983:Molybdoferredoxin; D014233:Tricarboxylic Acids; C028143:homocitric acid; D013455:Sulfur; D008982:Molybdenum; D007501:Iron; D009591:Nitrogenase