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Review
. 2014 Apr 23;114(8):4063-80.
doi: 10.1021/cr400463x. Epub 2013 Dec 13.

Biosynthesis of nitrogenase metalloclusters

Markus W Ribbe  1 , Yilin Hu, Keith O Hodgson, Britt Hedman
Affiliations
Review

Biosynthesis of nitrogenase metalloclusters

Markus W Ribbe et al. Chem Rev. .
No abstract available

PubMed Disclaimer

Conflict of interest statement

The author declares no competing financial interest.

Figures

Figure 1
Figure 1
Crystal structure of the ADP•AlF4−-stabilized NifH/NifDK complex (A) and the relative positions of components involved in the transfer of electrons (B). The two subunits of NifH are colored gray and light brown, and the α- and β-subunits of NifDK are colored red and light blue, respectively, in A. The same subunits 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, light purple; S, gold; Mo, brown; O, red; C, dark gray; N, dark blue; Mg, green; Al, beige; F, light blue. PYMOL was used to create this figure (PDB ID: 1N2C).
Figure 2
Figure 2
Crystal structures of the PN (A) and POX (B) states of the P-cluster and the M-cluster (C). The clusters are shown as ball-and-stick models. The atoms are shown as transparent balls and colored as those in Figure 1; and the ligands are shown as sticks. PYMOL was used to create this figure (PDB IDs: 1M1N and 3MIN). HC, homocitrate.
Figure 3
Figure 3
Biosynthesis of the M-cluster. This process is launched by NifS and NifU, which mobilize Fe and S for the sequential formation of [Fe2S2] and [Fe4S4] clusters. Subsequently, a pair of [Fe4S4] clusters (designated the K-cluster) are coupled into an [Fe8S9] cluster (designated the L-cluster) on NifB concomitant with the radical SAM-dependent carbide insertion. The L-cluster is then converted to a mature M-cluster on NifEN upon insertion of Mo and homocitrate (HC) by NifH, followed by the transfer of the M-cluster to its target location in NiDK. The permanent metal clusters are colored grey and distinguished by shape, with [Fe2S2] clusters shown as diamonds, [Fe4S4] clusters shown as cubes, and P-clusters shown as rounded rectangles. The transient cluster intermediates are colored in a yellow/orange tone. Solid arrows connect events of cluster transformation within the same protein; whereas dashed arrows indicate cluster transfer between proteins.
Figure 4
Figure 4
Schematic presentation (A), structural details (B) and the accompanying spectral changes (C) of the formation of L-cluster on NifB. (A and B) NifU supplies [Fe4S4] clusters to NifB, which forms an [Fe8S9] L-cluster through the coupling of the two [Fe4S4] modules of the K-cluster concomitant with the insertion of a sulfur atom and a carbon atom in a radical SAM-dependent process. The L-cluster is subsequently transferred to NifEN. The clusters and the SAM molecule are shown as ball-and-stick models, with the atoms colored as those in Figure 1. PYMOL was used to create this figure (PDB ID: 3PDI). (C) The K-cluster and the SAM-cluster collectively give rise to a SAM-responsive, S = 1/2 signal at g = 2.02, 1.95 and 1.90 in the dithionite-reduced state (upper); whereas the L-cluster displays a characteristic g = 1.94 signal in the IDS-oxidized state (lower).
Figure 5
Figure 5
Cleavage of SAM (A), formation of 5′-dAD (B) and incorporation of 14C into the L-cluster (C) by NifEN-B, and proposed mechanisms of carbon insertion by NifB during the process of L-cluster formation (D and E). (A) The HPLC elution profile of the cleavage products of SAM (i.e., SAH and 5′-dAH) upon incubation with NifEN-B and dithionite (trace 4). The elution profiles of SAM (trace 1), SAH (trace 2) and 5′-dAH (trace 3) standards are also shown in this figure. (B) LC-MS analysis showing the formation of 5′-dAD, along with 5′-dAH, upon incubation of [methyl-d3] SAM with NifEN-B. (C) Autoradiograph of the 14C-labeled L-cluster extracted from NifEN-B after incubated with [methyl-14C] SAM. (D and E) Both carbon insertion mechanisms involve hydrogen atom abstraction from a SAM-derived methyl group, radical-mediated methyl transfer to the K-cluster, and continued deprotonation till an interstitial carbide atom is formed in the center of the L-cluster. However, the initial step in D involves the transfer of methyl group via an SN2 mechanism, 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 S atom of the K-cluster; whereas the initial step in E involves the formation of a methyl radical via reductive cleavage of SAM, followed by the transfer of this transient intermediate to an Fe atom of the K-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 1. PYMOL was used to create this figure (PDB ID: 3PDI).
Figure 6
Figure 6
Schematic presentation (A), structural details (B) and the accompanying spectral changes (C and D) of the maturation of M-cluster on NifEN. (A and B) NifEN hosts the conversion of L- to M-cluster upon the replacement of a terminal Fe atom by Mo and homocitrate. NifH serves as an ATP-dependent Mo/HC insertase in this process. Following the completion of M-cluster assembly on NifEN, M-cluster is delivered to its destined location in NifDK. The permanent metal clusters are colored grey, with the [Fe4S4] clusters and the P-clusters shown as cubes and rounded rectangles, respectively. The transient cluster intermediates are colored in a yellow/orange tone. Mo, molybdenum; HC, homocitrate. Clusters are shown as ball-and-stick models, with the atoms colored as those in Figure 1. PYMOL was used to generate this figure (PDB IDs: 3PDI and 1M1N). (C and D) The L-cluster, along with the permanent [Fe4S4] clusters on NifEN, gives rise to a composite S = 1/2 signal at g = 2.09, 1.91 and 1.85 in the dithionite-reduced state (D, upper); however, this cluster can be recognized by a unique g = 1.94 signal in the IDS-oxidized state (C, upper). Upon maturation, the M-cluster on NifEN displays a small signal at g = 4.45, 3.96, 3.60 and 2.03 in the dithionite-reduced state (D, middle, left). The same g = 3.96 and g = 2.03 features of the M-cluster are also observed in the IDS-oxidized state (C, middle). The S = 1/2 signal in the dithionite-reduced state (D, middle, right) originates solely from the permanent [Fe4S4] clusters on NifEN, as the portion of the S = 1/2 signal that originates from the L-cluster disappears upon the conversion of L- to M-cluster. Upon delivery to NifDK, the M-cluster exhibits an S = 3/2 signal at g = 4.31, 3.67 and 2.01 in the dithionite-reduced (D, lower), and it is EPR silent in the IDS-oxidized state (C, lower).
Figure 7
Figure 7
Fe K-edge XAS spectra (A) of the L-cluster (blue) and the M-cluster (red) in the extracted state and Mo K-edge XAS spectra (B) of the M-clusters on NifEN (blue) and NifDK (red). A high degree of similarity is observed between the Fe K-edge XAS spectra of the L- and M-clusters and between the Mo K-edge XAS spectra of NifEN- and NifDK-bound M-clusters.
Figure 8
Figure 8
Schematic presentation (A) and the accompanying spectral changes (B and C) of the mobilization of molybdenum and homocitrate by NifH. (A) Molybdenum (Mo) and homocitrate (HC) can be “loaded” on NifH in the presence of ATP and reductant. Mo is reduced in this process (indicated as Moox→Mored in the figure), and it may enter NifH by attaching to the position that corresponds to the γ-phosphate of ATP upon ATP hydrolysis. Subsequently, the “loaded” NifH delivers Mo and HC to the NifEN-associated L-cluster and transforms it into a fully matured M-cluster (also see Figure 6). Mo is shown as a brown sphere; and homocitrate is shown as a ball-and-stick model. The [Fe4S4] cluster of NifH is represented by a gray cube. (B) Normalized Mo K-edge XAS spectra of molybdate (upper) and the Mo/HC-”loaded” NifH (lower). A reference line is drawn at 20,020 eV for the comparison of edge positions. (C) EPR spectra of the MgATP-bound NifH (upper) and the Mo/HC-”loaded” NifH (lower).
Figure 9
Figure 9
Transfer of the M-cluster between NifEN and NifDK (A), and ribbon diagrams (B) and surface presentations (C) of apo NifDK, NifEN and holo NifDK. (A) NifEN and NifDK share a common cluster insertion mechanism, which involves three sequential conformations: an apo conformation containing a cluster insertion path, an intermediary conformation upon docking of the cluster at the entrance of the path, and a holo conformation following the insertion of the cluster along the path into its binding site. The biosynthetic events on NifEN and NifDK are connected via complex formation and cluster transfer between the two proteins. The permanent clusters are colored grey, with the [Fe4S4] clusters on NifEN shown as cubes and the permanent P-clusters on NifDK shown as rounded rectangles. The transient clusters are colored in a yellow/orange tone. (B) Structures of the αβ-dimers of apo NifDK (upper), NifEN (middle) and holo NifDK (lower), showing the location of the M-cluster sites, as well as the L- and M-clusters, in the protein. All clusters are shown as space-filling models, with the atoms colored as those in Figure 1. The permanent clusters in NifEN ([Fe4S4] cluster) and NifDK (P-cluster) are rendered transparent in the background. (C) Electrostatic surface potentials of the αβ-dimers of apo-NifDK (upper), NifEN (middle) and holo-NifDK (lower), showing the locations of insertion paths in these proteins. Negative surface potentials are colored red, and positive surface potentials are colored blue. PYMOL was used to prepare this figure (PDB IDs: 1L5H, 3PDI and 1M1N).
Figure 10
Figure 10
Close-up of the M-cluster insertion path in apo NifDK. Some key residues for M-cluster insertion (illustrated as sticks) can be found along a positively charged insertion path that extends from the surface of the protein to the cluster-binding site within the protein. These residues include (1) the “lid loop” residue, Hisα362, which may provide the first docking point for the M-cluster at the entrance of the insertion path; (2) the “His triad” residues, Hisα274, Hisα442 and Hisα451, which may provide an intermediary docking point for the M-cluster halfway down the path; and (3) the “switch/lock” residues, Hisα442 and Trpα444, which may secure the FeMoco by the bulky side chain of Trpα444 through a switch in positions between the two residues at the end of the path. The electrostatic surface potentials of apo NifDK are colored as those in Figure 9.
Figure 11
Figure 11
Biosynthesis of the P-cluster. This process is launched by the actions of NifS and NifU, which mobilize Fe and S for the sequential formation of [Fe2S2] and [Fe4S4] clusters. Subsequently, a pair of [Fe4S4]-like clusters (designated the P*-cluster) are fused into an [Fe8S7] P-cluster at the α/β-subunit interface of NifDK. The two P-clusters in NifDK are assembled one at a time, which renders a stepwise assembly of the two αβ-dimers of this protein. The formation of the “first” P-cluster involves the sole action of NifH, whereas the formation of the “second” P-cluster requires the concerted action of NifH and NifZ. The maturation of the P-cluster not only provides added stability to each αβ-subunit interface, but also induces a conformational change that “opens” up the M-cluster site, thereby allowing the insertion of the M-cluster and the completion of the assembly process of NifDK. The sequential conformations of NifDK along the biosynthetic pathway are represented by three NifDK variants: (1) ΔnifH NifDK, which contains two [Fe4S4]-like cluster pairs, one per αβ-dimer; (2) ΔnifB ΔnifZ NifDK, which contains one P-cluster in one αβ-dimer and one [Fe4S4]-like cluster pair in the other αβ-dimer; and (3) ΔnifB NifDK, which contains two P-clusters, one per αβ-dimer. The P*-cluster is shown as a pair of grey cubes at the α/β-subunit interface. All other clusters are depicted as those in Figure 3. Solid arrows connect events of cluster transformation within the same protein, whereas dashed arrows indicate cluster transfer between proteins.
Figure 12
Figure 12
Structural details (A, B and C) and the accompanying spectral changes (D and E) of the coupling of a pair of [Fe4S4]-like clusters (designated the P*-cluster) into a mature [Fe8S7] P-cluster at the α/β-subunit interface of NifDK. (A and B) Ribbon diagrams of the αβ-dimers of the P*-cluster-containing ΔnifH NifDK (upper) and the P-cluster-containing ΔnifB NifDK (lower). The diagram on top represents the best model of ΔnifH NifDK based on the SAXS data, which was constructed from the structure of holo NifDK by deletion of the M-cluster, followed by a symmetric 6 Å-translation of the α- and β-subunits about the y axis. The subunits and atoms are colored as those in Figure 1, and the polypeptides are rendered transparent in B. PYMOL was used to create this figure (PDB IDs: 1M1N and 1L5H). (C) The P*-cluster comprises one standard [Fe4S4] cubane (upper) and one [Fe4S4]-like fragment that is distorted or coordinated by additional light atoms (lower), which can be reductively coupled into a [Fe8S7] structure upon incubation with NifH, MgATP and dithionite. The clusters are shown as ball-and-stick models, and the atoms are colored as those in Figure 1. (D and E) EPR spectra of the cluster species of ΔnifH NifDK in dithionite-reduced (D) and IDS-oxidized (E) states before (upper) and after (lower) maturation. Upon maturation, the S = 1/2 signal at g = 2.05, 1.93, and 1.90 (D, upper), which is characteristic of the P*-cluster, disappears; whereas the g = 11.8 parallel-mode signal (E, lower), which is characteristic of the mature P-cluster in the POX state, emerges.
Figure 13
Figure 13
Time-dependent increase of the relative area of the POX-specific, g = 11.8 parallel-mode EPR signal (A), the relative specific activities (B) and the XAS/EXAFS-derived ratio between short and long Fe-Fe distances (C) of ΔnifH NifDK (blue) and ΔnifBΔnifZ NifDK (red). The horizontal dashed lines (A, B and C) represent the assembly of the “first” P-cluster in NifDK; whereas the vertical dashed lines (A and B) mark the starting points for the alignment of ΔnifH NifDK and ΔnifBΔnifZ NifDK data.
Figure 14
Figure 14
Flow diagrams of the “in situ” pathway of P-cluster assembly (illustrated in the orange block) and the “ex situ” pathway of M-cluster assembly (illustrated in the light blue block). Biosyntheses of the P- and M-clusters share two common apparatus, NifS and NifU, at the early stages of these processes, where [Fe4S4] units are generated as the building blocks for the further construction of these clusters. Both pathways then use a pair of 4Fe units for the formation of an 8Fe cluster; however, this step occurs on different proteins in these pathways. In the case of P-cluster assembly, a pair of [Fe4S4]-like clusters (P*-cluster) are coupled into an [Fe8S7] cluster (P-cluster) on NifDK in a process that requires the actions of NifH and NifZ; whereas in the case of M-cluster assembly, two [Fe4S4] modules (K-cluster) are coupled into an [Fe8S9] cluster (L-cluster) on NifB concomitant with the radical SAM-dependent C insertion and the addition of S in an unknown mechanism. The L-cluster is further processed on NifEN into a [MoFe7S9C-homocitrate] cluster (M-cluster) upon NifH-mediated insertion of Mo and homocitrate. All clusters are shown as ball-and-stick models, with the atoms colored as those in Figure 1. PYMOL was used to create this figure (PDB IDs: 1M1N and 3PDI).
See this image and copyright information in PMC

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