Tracing the ‘ninth sulfur’ of the nitrogenase cofactor via a semi-synthetic approach

Abstract

The M-cluster is the [(homocitrate)MoFe7S9C] active site of nitrogenase that is derived from an 8Fe core assembled via coupling and rearrangement of two [Fe4S4] clusters concomitant with the insertion of an interstitial carbon and a ‘9th sulfur’. Combining synthetic [Fe4S4] clusters with an assembly protein template, here we show that sulfite can give rise to the ‘9th sulfur’ that is incorporated in the catalytically important belt region of the cofactor after the radical SAM-dependent carbide insertion and the concurrent 8Fe-core rearrangement have already taken place. Based on the differential reactivity of the formed cluster species, we also propose a new [Fe8S8C] cluster intermediate, the L*-cluster, that is similar to the [Fe8S9C] L- cluster but lacks the ‘9th S’ from sulfite. This work provides a semi-synthetic tool for protein reconstitution that could be widely applicable for the functional analysis of other FeS systems.

Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB

“…A member of the radical S-adenosyl-L-methionine (SAM) enzyme family 7,8 , NifB is a monomeric protein carrying three [Fe 4 S 4 ] clusters: [9][10][11][12] one, designated the SAM-cluster, is ligated by a canonical CxxxCxxC motif and coordinates the SAM-binding [Fe 4 S 4 ] cluster; the other two, designated the K-cluster, are ligated by a number of conserved ligands and supply the full complement of iron (Fe) atoms for the biosynthesis of an 8Fe precursor to the mature cofactor. Previous studies of the NifB proteins from Azotobacter vinelandii and Methanosarcina acetivorans have led to the proposal that NifB utilizes a unique, radical SAM-dependent mechanism for carbide insertion concomitant with the transformation of the K-cluster to an [Fe 8 S 9 C] cluster (designated the Lcluster), which represents both an 8Fe precursor and an all-Fe core of the M-cluster [13][14][15][16] . This process begins with an S N 2-type transfer of a methyl group from a SAM molecule to the K-cluster (Supplementary Fig.…”

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN

“…Designated the M-cluster (or FeMoco), the cofactor of Mo-nitrogenase is ac omplex [(Rhomocitrate)MoFe 7 S 9 C] cluster [6][7][8] that is unique in biology and unprecedented in chemistry.T he biosynthesis of the Mcluster ( Figure 1a) [9,10] is ah ighly complex process and encompasses all components required for the assembly of (designated the L-cluster) concomitant with the insertion of aS AM-derived interstitial carbide and as ulfite-derived "9 th sulfur". [11,12] An all-iron analogue of the M-cluster,t he Lcluster,isthen transferred to the biosynthesis scaffold NifEN, where it is matured into an M-cluster upon ATP-dependent insertion of Mo and homocitrate by NifH. [13] This event is followed by the insertion of the M-cluster into its target binding site in apo-NifDK, leading to the formation of ac atalytically competent holo-NifDK protein.…”

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN

“…It begins with the concerted action of NifS and NifU, which mobilize Fe and S for the synthesis of [Fe 4 S 4 ] clusters. Subsequently, a pair of [Fe 4 S 4 ] clusters (designated the K‐cluster) are delivered to NifB, a radical S ‐adenosyl‐ l ‐methionine (SAM) enzyme, where they are coupled and rearranged into an [Fe 8 S 9 C] cluster (designated the L‐cluster) concomitant with the insertion of a SAM‐derived interstitial carbide and a sulfite‐derived “9 th sulfur” . An all‐iron analogue of the M‐cluster, the L‐cluster, is then transferred to the biosynthesis scaffold NifEN, where it is matured into an M‐cluster upon ATP‐dependent insertion of Mo and homocitrate by NifH .…”