Radical SAM-dependent carbon insertion into the nitrogenase M-cluster

Abstract

The active site of nitrogenase, the M-cluster, is a metal-sulfur cluster containing a carbide at its core. Using radiolabeling experiments, we show that this carbide originates from the methyl group of S-adenosylmethionine (SAM) and that it is inserted into the M-cluster by the assembly protein NifB. Our SAM cleavage and deuterium substitution analyses suggest a similarity between the mechanism of carbon insertion by NifB and the proposed mechanism of RNA methylation by the radical SAM enzymes RlmN and Cfr, which involves methyl transfer from one SAM equivalent, followed by hydrogen atom abstraction from the methyl group by a 5'-deoxyadenosyl radical generated from a second SAM equivalent. This work is an initial step toward unraveling the importance of the interstitial carbide and providing insights into the nitrogenase mechanism.

Fig. 1

(A) Biosynthesis of M-cluster on NifEN-B fusion protein, which involves SAM-dependent conversion of K-cluster to L-cluster on NifB, transfer of L-cluster to NifEN, maturation of L-cluster upon NifH-mediated insertion of Mo and homocitrate, and transfer of the resultant M-cluster to NifDK. (B) Coupling of the 4Fe units of K-cluster into an 8Fe L-cluster via carbon- and sulfur-insertion, and conversion of the L-cluster to a mature M-cluster upon insertion of Mo and HC (homocitrate). The L-cluster represents an all-iron homolog of the M-cluster.

Fig. 2

(A) HPLC elution profiles of SAM in the (1) absence and (2) presence of NifEN-B and dithionite, compared with those of (3) SAH and (4) 5′-dAH standards. (B) LC-MS analysis of 5′-dAH recovered after incubation with unlabeled SAM (left) or [methyl-d₃] SAM (right). The ratio of 5′-dAH to SAH produced in the presence of NifEN-B was 1.8 ± 0.1 (N=5). In a maturation assay, the yields of SAH and M-cluster were 2.0 ± 0.2 and 1.7 ± 0.1 μM, respectively, in the presence of 10 μM SAM and 10 μM NifEN-B (N=5).

Fig. 3

(A) Protein fractions captured on affinity (IMAC) and anion-exchange (DEAE) resins after incubation of [methyl-¹⁴C]-SAM with (1) His-tagged NifEN-B alone; (2) His-tagged NifEN-B, non-tagged NifH, and His-tagged NifDK; (3) His-tagged NifEN-B, non-tagged NifH, and non-tagged NifDK; and (4) His-tagged NifEN-B, His-tagged NifH, and non-tagged NifDK. Assays 2-4 also contained dithionite, ATP, molybdate, and homocitrate. (B) Experiments (B, 1-4) were identical to those in (A, 1-4), except that [carboxyl-¹⁴C]-SAM was used instead of [methyl-¹⁴C]-SAM. Experiment (A, 4) was included as a positive control here (B, 5). (C) Clusters extracted from (1) the IMAC fraction in (A, 1); and (2) the IMAC fraction (left) and DEAE (right) fraction in (A, 4). (D) Activities of C₂H₂ reduction by extracted clusters alone (1-3), after maturation and transfer of clusters to apo NifDK (4, 5), or upon direct transfer of cluster to apo NifDK (6). Clusters used for activity analyses were from C1, left (1, 4); C2, left (2, 5); and C2, middle (3, 6), respectively. Activities of clusters upon maturation and/or incorporation into apo NifDK (4-6) are indicated by *.

Fig. 4

Proposed mechanisms of carbide insertion involve (A) transfer of a methyl group from SAM to the 4Fe K-cluster via an SN2 mechanism, followed by reductive cleavage of a second equivalent of SAM to generate 5-dA•, which abstracts a hydrogen atom from the methyl group on the K-cluster; or (B) reductive cleavage of a methyl group from SAM, followed by transfer of the resultant methyl radical to the 4Fe K-cluster and abstraction of a hydrogen atom of this group by 5-dA•. Continued deprotonation of the C intermediate in A or B must occur concomitant with its incorporation into the 8Fe L-cluster as an interstitial carbide atom. Atoms of the clusters are colored as follows: Fe, orange; S, yellow; C, grey.