NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway.
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.
Hernandez
Jose A
JA
Department of Plant and Microbial Biology, University of California-Berkeley, Berkeley, CA 94720, USA.
Igarashi
Robert Y
RY
Soboh
Basem
B
Curatti
Leonardo
L
Dean
Dennis R
DR
Ludden
Paul W
PW
Rubio
Luis M
LM
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*
D001426:Bacterial Proteins; D058085:Iron Compounds; D008983:Molybdoferredoxin; C085381:NifB cofactor; C118795:NifX protein, Bacteria