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In nitrogenase biosynthesis, the iron-molybdenum cofactor (FeMo-co) is externally assembled at scaffold proteins and delivered to the NifDK nitrogenase component by the NafY metallochaperone. Here we have used nuclear magnetic resonance, molecular dynamics, and functional analysis to elucidate the environment and coordination of FeMo-co in NafY. H 121 stands as the key FeMo-co ligand. Regions near FeMo-co diverge from H 121 and include the η1, α1, α2 helical lobe and a narrow path between H 121 and C 196 ., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The nitrogenase MoFe protein contains two different FeS centers, the P-cluster and the iron-molybdenum cofactor (FeMo-co). The former is a [Fe 8 S 7 ] center responsible for conveying electrons to the latter, a [MoFe 7 S 9 C-( R )-homocitrate] species, where N 2 reduction takes place. NifB is arguably the key enzyme in FeMo-co assembly as it catalyzes the fusion of two [Fe 4 S 4 ] clusters and the insertion of carbide and sulfide ions to build NifB-co, a [Fe 8 S 9 C] precursor to FeMo-co. Recently, two crystal structures of NifB proteins were reported, one containing two out of three [Fe 4 S 4 ] clusters coordinated by the protein which is likely to correspond to an early stage of the reaction mechanism. The other one was fully complemented with the three [Fe 4 S 4 ] clusters (RS, K1 and K2), but was obtained at lower resolution and a satisfactory model was not obtained. Here we report improved processing of this crystallographic data. At odds with what was previously reported, this structure contains a unique [Fe 8 S 8 ] cluster, likely to be a NifB-co precursor resulting from the fusion of K1- and K2-clusters. Strikingly, this new [Fe 8 S 8 ] cluster has both a structure and coordination sphere geometry reminiscent of the fully reduced P-cluster (P N -state) with an additional μ 2 -bridging sulfide ion pointing toward the RS cluster. Comparison of available NifB structures further unveils the plasticity of this protein and suggests how ligand reorganization would accommodate cluster loading and fusion in the time-course of NifB-co synthesis., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Mo and Fe K-edge EXAFS analysis of NifQ shows the presence of a [MoFe 3 S 4 ] cluster and a second independent Mo environment that includes Mo-O bonds and Mo-S bonds. Both environments are relevant to FeMo-co biosynthesis and may represent different stages of Mo biochemical transformations catalyzed by NifQ.