Purification of a NifEN protein complex that contains bound molybdenum and a FeMo-Co precursor from an Azotobacter vinelandii DeltanifHDK strain.
The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. A His-tagged version of the NifEN complex has been previously purified and shown to carry two identical [4Fe-4S] clusters of unknown function and a [Fe-S]-containing FeMo-co precursor. We have improved the purification of the his-NifEN protein from a DeltanifHDK strain of Azotobacter vinelandii and have found that the amounts of iron and molybdenum within NifEN were significantly higher than those reported previously. In an in vitro FeMo-co synthesis system with purified components, the NifEN protein served as a source of both molybdenum and a [Fe-S]-containing FeMo-co precursor, showing significant FeMo-co synthesis activity in the absence of externally added molybdate. Thus, the NifEN scaffold protein, purified from DeltanifHDK background, contained the Nif-Bco-derived Fe-S cluster and molybdenum, although these FeMo-co constituents were present at different levels within the protein complex.
Soboh
Basem
B
Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, California 94720, USA.
Igarashi
Robert Y
RY
Hernandez
Jose A
JA
Rubio
Luis M
LM
D016948:Azotobacter vinelandii / Q000378:metabolism; D002021:Buffers; D002352:Carrier Proteins / Q000737:chemistry; D002846:Chromatography, Affinity; D003035:Cobalt / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008982:Molybdenum / Q000737:chemistry*; D008983:Molybdoferredoxin / Q000737:chemistry*; D009591:Nitrogenase / Q000737:chemistry; D011485:Protein Binding; D015032:Zinc / Q000737:chemistry
D002021:Buffers; D002352:Carrier Proteins; D008670:Metals; D008983:Molybdoferredoxin; C044659:molybdate; D003035:Cobalt; D008982:Molybdenum; D007501:Iron; D009591:Nitrogenase; D015032:Zinc