An unexpected P-cluster like intermediate en route to the nitrogenase FeMo-co

Abstract

The nitrogenase MoFe protein contains two different FeS centers, the P-cluster and the iron-molybdenum cofactor (FeMo-co). The former is a [Fe₈S₇] center responsible for conveying electrons to the latter, a [MoFe₇S₉C-(R)-homocitrate] species, where N₂ reduction takes place. NifB is arguably the key enzyme in FeMo-co assembly as it catalyzes the fusion of two [Fe₄S₄] clusters and the insertion of carbide and sulfide ions to build NifB-co, a [Fe₈S₉C] precursor to FeMo-co. Recently, two crystal structures of NifB proteins were reported, one containing two out of three [Fe₄S₄] 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 [Fe₄S₄] 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 [Fe₈S₈] cluster, likely to be a NifB-co precursor resulting from the fusion of K1- and K2-clusters. Strikingly, this new [Fe₈S₈] cluster has both a structure and coordination sphere geometry reminiscent of the fully reduced P-cluster (P^N-state) with an additional μ²-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.

Fig. 1. MthNifB crystal structure. The radical SAM core structure β-barrel is depicted in red (strands) and blue (helices). The N- and C-terminal domains are depicted in orange and green, respectively. The FeS-cluster iron and sulfur atoms are represented in brown and yellow spheres, respectively. The bridging cysteine residue C¹⁸ that belongs to the N-terminal domain is indicated. The flexible loop is depicted in purple. C⁵² and E⁵³ are also indicated.

Fig. 2. Close-up of the K1 and K2 cluster-binding site. (A) F_o − F_c difference Fourier electron density map (omit map) around the K-cluster contoured at 3σ. (B) Coordination geometry of the K-cluster in the newly refined structure. (C) Model of two independent [Fe₄S₄] clusters in the same F_o − F_c difference Fourier electron density map (omit map) as in (A), illustrating a lower fit when compared to (A). The arrow indicates the position of the bump observed in the electron density map (see also Fig. S2C‡) (D) coordination geometry obtained when refining two independent [Fe₄S₄] clusters. Both the ligand coordination and the [Fe₄S₄] cluster contacts are chemically meaningless. Stereoviews of each panel are available as Fig. S7–S10, respectively.

Fig. 3. Overlay of the P-cluster from the MoFe protein (P^N-state; PDB 3U7Q) and the K-cluster from NifB. Only the P- and K-cluster atoms were used for superposition. The carbon atoms of amino acid residues serving as ligands of the P- and K-clusters are depicted in black and purple, respectively. In the MoFe-protein, residues C⁶², C⁸⁸ and C¹⁵⁴ belong to chain C (NifD or α chain), while residues C⁷⁰, C⁹⁵ and C¹⁵³ belong to chain D (NifK or β chain). A stereoview is available as Fig. S11.

Fig. 4. Schematic view of the early steps for NifB-co formation by NifB. (A) NifB initially binds two [Fe₄S₄] clusters, an RS-cluster (not shown) and a K1-cluster with four ligands including labile E⁵³ on a flexible loop. (B) E⁵³ is displaced allowing a second [Fe₄S₄] cluster, K2, to bind to previously disordered C-terminal residues C²⁶⁰, C²⁶³ and likely one or two unknown additional ligands. (C) The ordered coordination environment then forces K1 and K2 into proximity where they fuse into a single [Fe₈S₈] K-cluster (the structure in this work). NB: This mechanism is not charge balanced, pending further spectroscopic evidence to identify the oxidation states of the clusters.