A journey into the active center of nitrogenase

Abstract

Nitrogenase catalyzes the reduction of N2 to NH3, a key step in the global nitrogen cycle. This article describes our journey toward the definition of a complete molecular structure of the active site of nitrogenase, with an emphasis on the discovery of the interstitial carbide and the radical SAM-dependent insertion of this atom into the active FeMo cofactor site of nitrogenase.

Fig. 1

Structural models of the molybdenum coordination environment in the FeMo cofactor based on Mo K-edge XAS/EXAFS analysis in 1978.

Fig. 2

Crystal structure of the ADP•AlF₄⁻-stabilized Fe protein/MoFe protein complex (A) and the relative positions of components involved in electron transfer during catalysis (B). One “catalytic unit”, which consists of one αβ-half of the complex, is shown. The two subunits of the Fe protein are colored gray and light brown, respectively, and the α- and β-subunits of the MoFe protein are colored red and light blue, respectively, in A. The protein peptides of one αβ-dimer are rendered transparent in the background in B. All clusters and ADP•AlF₄⁻ are shown as space-filling models. Atoms are colored as follows: Fe, orange; S, yellow; Mo, cyan; O, red; C, gray; N, blue; Mg, dark green; Al, light green; F, light blue. PYMOL was used to create this figure (PDB ID: 1N2C).

Fig. 3

Structure of the FeMo cofactor based on data from 1992 (A), 2002 (B), and 2011 (C). The atoms are colored as those in Figure 2. His^α442 and Cys^α275, the two protein ligands of the FeMo cofactor, are indicated. HC, homocitrate. PYMOL was used to create this figure (PDB IDs: 1N2C, 1M1N, 3U7Q).

Fig. 4

Biosynthesis of the FeMo cofactor. Shown are the coupling of two [Fe₄S₄] clusters into a [Fe₈S₉] precursor concomitant with carbide insertion (on NifB) and the subsequent conversion of the precursor to a fully matured FeMo cofactor upon Fe protein-mediated insertion of Mo and homocitrate (on NifEN).

Fig. 5

Proposed pathways of carbon insertion by NifB into the precursor of the FeMo cofactor. Both pathways involve hydrogen atom abstraction from a SAM-derived methyl group, radical-mediated methyl transfer to a [Fe₄S₄] cluster, and continued deprotonation till an interstitial carbide ion is formed in the center of the precursor. However, the pathway in A begins with the transfer of methyl group via an SN2 mechanism, and it is followed by the formation of a methylene radical upon hydrogen atom abstraction by 5′-dA• and the subsequent transfer of this radical intermediate to a sulfur atom of the [Fe₄S₄] cluster; whereas the pathway in B begins the formation of a methyl radical via reductive cleavage of SAM, and it is followed by the transfer of this transient intermediate to an iron atom of the [Fe₄S₄] cluster and the subsequent processing of this intermediate into a methylene radical. The clusters are shown as ball-and-stick models, with the atoms colored as those in Figure 2. PYMOL was used to create this figure (PDB ID: 3PDI).