title: NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway.
abstract: The iron-molybdenum cofactor of nitrogenase (FeMo-co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre-synthesized apo-dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo-co precursors or FeMo-co during cofactor synthesis. In this work, the binding of FeMo-co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo-co synthesis. Purified NifX binds NifB cofactor (NifB-co), a precursor to FeMo-co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe-S] cluster that serves as a FeMo-co precursor, and we have designated it as the VK-cluster. In contrast to NifB-co, the VK-cluster is electronic paramagnetic resonance (EPR)-active in the reduced and the oxidized states. The NifX/VK-cluster complex is unable to support in vitro FeMo-co synthesis in the absence of NifEN because further processing of the VK-cluster into FeMo-co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo-co precursors and thus help control their flux during FeMo-co synthesis.
authors.affiliation: Department of Plant and Microbial Biology, University of California-Berkeley, Berkeley, CA 94720, USA.
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: Leonardo
authors.initials: L
authors.lastname: Curatti
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Dean
authors.forename: Paul W
authors.initials: PW
authors.lastname: Ludden
authors.forename: Luis M
authors.initials: LM
authors.lastname: Rubio
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D053898:Biosynthetic Pathways; D005798:Genes, Bacterial; D058085:Iron Compounds / Q000378:metabolism*; D008983:Molybdoferredoxin / Q000096:biosynthesis* / Q000737:chemistry; D009586:Nitrogen Fixation / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D058085:Iron Compounds; D008983:Molybdoferredoxin; C085381:NifB cofactor; C118795:NifX protein, Bacteria