Requirement of NifX and Other Nif Proteins for in vitro Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Abstract

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATPregenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42.48 (⌬nifENX ⌬vnfE) results in a 12-to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.

Chlorobaculum tepidum TLS Displays a Complex Transcriptional Response to Sulfide Addition

“…MTH1175 has no known function, but members of the two closely related subfamilies (NifB [cd00852] and NifX [cd00853]) have been shown to have Fe-S cluster binding abilities and to be involved in biosynthesis of the FeMo cofactor involved in nitrogen fixation (51). Another member of the MTH1175 subfamily, sulfur-induced protein A (sipA), was recently found to be expressed in response to elemental sulfur addition in Pyrococcus furiosus (52,53).…”

VnfY Is Required for Full Activity of the Vanadium-Containing Dinitrogenase in Azotobacter vinelandii

“…In addition, the nifB gene, and another gene of unknown function that bears primary sequence similarity to the nifB gene, also exhibits some primary sequence similarity to the nifX gene. The involvement of this family of proteins in the biosynthesis of FeMo-co or FeV-co was suggested based on the ability of certain of them to bind to FeMo-co, FeV-co, or their precursors and on the capacity of NifX to stimulate the in vitro synthesis of FeMo-co threefold (19). It has been also proposed that NafY and NifY are involved in the insertion of FeMo-co into the nif apodinitrogenase (7,8).…”

The NifX Protein is Involved in the Final Stages of FeMo-cofactor Transport to the MoFe Protein

“…The small molecule binding domain (SMBD) of NifX has widespread phyletic distribution and therefore indicates an ancient origin or extensive dissemination by horizontal transfer during evolution, or both [33] . Eventhough a deletion in the nifX gene did not show a significant effect on the nitrogenase activity in vivo, the purified NifX protein was shown to be important in the stimulation of FeMoco synthesis in an in vitro FeMoco synthesis reaction [10,34] . The NifX, NifB, NifY and NafY are most likely related proteins, owing to their overall sequence similarity and the presence of a FeMoco binding 'core domain' in all of them [35] .…”

“…Considerable research efforts have been directed towards understanding the biosynthesis and sequential assembly of the FeMoco and it is known that various nitrogen fixation (nif) genes play a role in the biosynthesis, assembly, transport and insertion of the FeMocofactor [7,8] . The products of the nifB, nifV, nifQ, nifH, nifE, nifN, nifX and nafY genes have been identified to be the prime players in these processes [9][10][11] .…”

“…In NifB, the NIFX SMBD is fused with a biotin-synthase-like, metal-dependent catalytic domain and in the archaeal MTH1172 protein, it is fused to a cation transporter [33] . Previous studies in Azotobacter vinelandii have identified certain important molecular interactions of the NifX protein with the other Nif proteins involved in the FeMoco biosynthesis/transport pathway; mainly it was shown that NifX binds to the NifB-co and also a FeMoco precursor from the NifEN complex [10,30] . Clearly, a protein-protein interaction based study of NifX with other FeMoco maturation proteins would enhance our insight into the role of NifX and may help to solve unanswered questions regarding its position in the FeMoco biosynthetic pathway.…”