Assembly scaffold NifEN: A structural and functional homolog of the nitrogenase catalytic component

Abstract

NifEN is a biosynthetic scaffold for the cofactor of Mo-nitrogenase (designated the M-cluster). Previous studies have revealed the sequence and structural homology between NifEN and NifDK, the catalytic component of nitrogenase. However, direct proof for the functional homology between the two proteins has remained elusive. Here we show that, upon maturation of a cofactor precursor (designated the L-cluster) on NifEN, the cluster species extracted from NifEN is spectroscopically equivalent and functionally interchangeable with the native M-cluster extracted from NifDK. Both extracted clusters display nearly indistinguishable EPR features, X-ray absorption spectroscopy/extended X-ray absorption fine structure (XAS/EXAFS) spectra and reconstitution activities, firmly establishing the M-cluster-bound NifEN (designated NifEN M ) as the only protein other than NifDK to house the unique nitrogenase cofactor. Iron chelation experiments demonstrate a relocation of the cluster from the surface to its binding site within NifEN M upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifEN M with an M-cluster in one αβ-half and an empty clusterbinding site in the other αβ-half, which led to the proposal of a stepwise assembly mechanism of the M-cluster in the two αβ-dimers of NifEN. Perhaps most importantly, NifEN M displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster-bound αβ-dimer of NifEN M as a structural and functional mimic of one catalytic αβ-half of NifDK while suggesting the potential of this protein as a useful tool for further investigations of the mechanistic details of nitrogenase.nitrogenase | catalysis | assembly | functional homolog | NifEN N itrogenase plays a key role in global nitrogen cycle through its unique ability to reduce nitrogen to ammonia under ambient conditions. The best characterized, Mo-dependent nitrogenase uses a two-component system for substrate reduction, in which a specific reductase (designated the Fe protein) serves as the obligate electron donor for a catalytic component (designated the MoFe protein) during catalysis (1). The Fe protein (or NifH) is a γ 2 -dimer, which contains a [Fe 4 S 4 ] cluster between the two subunits and an ATP-binding site within each subunit, whereas the MoFe protein (or NifDK) is an α 2 β 2 -tetramer, which contains a P-cluster ([Fe 8 S 7 ]) between each α/β-subunit interface and an M-cluster (also termed the cofactor; [MoFe 7 S 9 C-homocitrate]) within each α-subunit (2-4). Catalysis of nitrogenase involves formation of a functional complex between the two component proteins of the Mo-nitrogenase (5), which permits ATP-dependent transfer of electrons from the [Fe 4 S 4 ] cluster of NifH, via the P-cluster, to the M-cluster of NifDK and the subsequent reduction of substrate at the M-cluster site upon accumulation of a sufficient amount of electrons.Arguably the most complex metallocluster ...

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN

“…Consistent with sequence‐based predictions, crystallographic analysis confirmed an overall structural homology between NifEN and NifDK, showing an α 2 β 2 ‐tetrameric structure of NifEN with a [Fe 4 S 4 ] cluster bridged at each α/β‐subunit interface . Biochemical and spectroscopic studies further illustrated the transient nature of the cofactor‐binding site in NifEN, showing differences of this site among three A. vinelandii NifEN species generated under in vivo or in vitro conditions: one, designated NifEN, is free of any cofactor species; another, designated NifEN L , contains an L‐cluster; the third, designated NifEN M , contains an M‐cluster (Figure b) . These protein species represent different conformations of NifEN that appear sequentially during the cofactor assembly process: the cofactor‐free NifEN appears first in the process, and it can be readily converted to NifEN L upon incorporation of the L‐cluster, followed by conversion of NifEN L to NifEN M upon maturation of the L‐cluster to an M‐cluster.…”

“…Subsequently, NifEN M can serve as a cofactor donor for apo‐NifDK via direct protein–protein interactions, resulting in an active holo‐NifDK. Interestingly, results from metal chelation experiments point to a conformational change of NifEN that allows the cluster to be transferred from the protein surface to a cofactor‐binding site within the protein upon maturation of the L‐cluster to an M‐cluster . Crystallographic analyses further suggest the presence of a cofactor‐binding site in NifEN that is analogous to the cofactor‐binding site in NifDK.…”

Synthetic Analogues of Nitrogenase Metallocofactors: Challenges and Developments

“…Although progress towards N 2 reduction with synthetic Fe−S clusters has been limited, nitrogenase is known to reduce a variety of substrates other than N 2 . Extracted M‐, V‐ and L ‐clusters all demonstrate competency in the reduction of C 1 ‐compounds such as CN − , CO, and CO 2 , converting the substrates into short‐chain hydrocarbon products . These Fischer–Tropsch‐like reactions have been performed in both buffered aqueous and organic solvents, using [Eu II DTPA] 3− (DTPA=diethylenetriaminepentaacetate) or SmI 2 , respectively, as reductants .…”

Heterologous Expression and Engineering of the Nitrogenase Cofactor Biosynthesis Scaffold NifEN

“…Interestingly,r esults from metal chelation experiments point to ac onformational change of NifEN that allows the cluster to be transferred from the protein surface to acofactor-binding site within the protein upon maturation of the L-cluster to an M-cluster. [17] Crystallographic analyses further suggest the presence of ac ofactor-binding site in NifEN that is analogous to the cofactor-binding site in NifDK. In the case of NifEN,t he absence of certain key residues (namely,H is 442 and Tr p 444 of NifDK), which presumably change positions upon M-cluster insertion and thereby "lock" the M-cluster in place,a long with substitution of the Mo ligand (namely,His 442 of NifDK) by Asn and replacement of the homocitrate ligand (namely, Lys 426 of NifDK) by Arg, allows the M-cluster to be released from NifEN and transferred to NifDK upon docking between the two proteins (Supporting Information, Figures S1 and S2).…”

“…[16] Biochemical and spectroscopic studies further illustrated the transient nature of the cofactorbinding site in NifEN,showing differences of this site among three A. vinelandii NifEN species generated under in vivo or in vitro conditions:o ne,d esignated NifEN,i sf ree of any cofactor species;another,designated NifEN L ,contains an Lcluster;t he third, designated NifEN M ,c ontains an M-cluster ( Figure 1b). [17] These protein species represent different conformations of NifEN that appear sequentially during the cofactor assembly process:t he cofactor-free NifEN appears first in the process,and it can be readily converted to NifEN L upon incorporation of the L-cluster,f ollowed by conversion of NifEN L to NifEN M upon maturation of the L-cluster to an M-cluster.S ubsequently,N ifEN M can serve as ac ofactor donor for apo-NifDK via direct protein-protein interactions, resulting in an active holo-NifDK. Interestingly,r esults from metal chelation experiments point to ac onformational change of NifEN that allows the cluster to be transferred from the protein surface to acofactor-binding site within the protein upon maturation of the L-cluster to an M-cluster.…”