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NifX and NifEN exchange NifB cofactor and the VK‐cluster, a newly isolated intermediate of the iron‐molybdenum cofactor biosynthetic pathway
Author(s) -
Hernandez Jose A.,
Igarashi Robert Y.,
Soboh Basem,
Curatti Leonardo,
Dean Dennis R.,
Ludden Paul W.,
Rubio Luis M.
Publication year - 2007
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2006.05514.x
Subject(s) - azotobacter vinelandii , nitrogenase , cofactor , iron–sulfur cluster , electron paramagnetic resonance , biology , biosynthesis , stereochemistry , biochemistry , crystallography , chemistry , enzyme , bacteria , physics , genetics , nitrogen fixation , nuclear magnetic resonance
Summary 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.