title: Formation of a homocitrate-free iron-molybdenum cluster on NifEN: implications for the role of homocitrate in nitrogenase assembly.
abstract: Molybdenum (Mo)-dependent nitrogenase is a complex metalloprotein that catalyzes the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) at the molybdenum-iron cofactor (FeMoco) site of its molybdenum-iron (MoFe) protein component. Here we report the formation of a homocitrate-free, iron-molybdenum ("FeMo") cluster on the biosynthetic scaffold of FeMoco, NifEN. Such a NifEN-associated "FeMo" cluster exhibits EPR features similar to those of the NifEN-associated, fully-complemented "FeMoco", which originate from the presence of Mo in both cluster species; however, "FeMo" cluster and "FeMoco" display different temperature-dependent changes in the line shape and the signal intensity of their respective EPR features, which reflect the impact of homocitrate on the redox properties of these clusters. XAS/EXAFS analysis reveals that the Mo centers in both "FeMo" cluster and "FeMoco" are present in a similar coordination environment, although Mo in "FeMo" cluster is more loosely coordinated as compared to that in "FeMoco" with respect to the Mo-O distances in the cluster, likely due to the absence of homocitrate that normally serves as an additional ligand for the Mo in the cluster. Subsequent biochemical investigation of the "FeMo" cluster not only facilitates the determination of the sequence of events in the mobilization of Mo and homocitrate during FeMoco maturation, but also permits the examination of the role of homocitrate in the transfer of FeMoco between NifEN and MoFe protein. Combined outcome of these studies establishes a platform for future structural analysis of the interactions between NifEN and MoFe protein, which will provide useful insights into the mechanism of cluster transfer between the two proteins.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.
authors.forename: Aaron Wolfe
authors.initials: AW
authors.lastname: Fay
authors.forename: Michael Aaron
authors.initials: MA
authors.lastname: Blank
authors.forename: Janice Mariko
authors.initials: JM
authors.lastname: Yoshizawa
authors.forename: Chi Chung
authors.initials: CC
authors.lastname: Lee
authors.forename: Jared Andrew
authors.initials: JA
authors.lastname: Wiig
authors.forename: Yilin
authors.initials: Y
authors.lastname: Hu
authors.forename: Keith Owen
authors.initials: KO
authors.lastname: Hodgson
authors.forename: Britt
authors.initials: B
authors.lastname: Hedman
authors.forename: Markus Walter
authors.initials: MW
authors.lastname: Ribbe
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry*; D008982:Molybdenum / Q000737:chemistry*; D008983:Molybdoferredoxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009586:Nitrogen Fixation; D009591:Nitrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014233:Tricarboxylic Acids / Q000737:chemistry*; D056928:X-Ray Absorption Spectroscopy
chemical_list: D001426:Bacterial Proteins; D008983:Molybdoferredoxin; D014233:Tricarboxylic Acids; C028143:homocitric acid; D008982:Molybdenum; D007501:Iron; D009591:Nitrogenase