FeMo-co Biosynthesis Research Articles

Molecular insights into nitrogenase FeMo cofactor insertion: the role of His 362 of the MoFe protein α subunit in FeMo cofactor incorporation

The assembly of the complex iron-molybdenum cofactor (FeMoco) of nitrogenase molybdenum-iron (MoFe) protein has served as one of the central topics in the field of bioinorganic chemistry for decades. Here we examine the role of a MoFe protein residue (His alpha362) in FeMoco insertion, the final step of FeMoco biosynthesis where FeMoco is incorporated into its binding site in the MoFe protein. Our data from combined metal, activity and electron paramagnetic resonance analyses show that mutations of His alpha362 to small uncharged Ala or negatively charged Asp result in significantly reduced FeMoco accumulation in MoFe protein, indicating that His alpha362 plays a key role in the process of FeMoco insertion. Given the strategic location of His alpha362 at the entry point of the FeMoco insertion funnel, this residue may serve as one of the initial docking points for FeMoco insertion through transient ligand coordination and/or electrostatic interaction.

Purification of a NifEN Protein Complex That Contains Bound Molybdenum and a FeMo-Co Precursor from an Azotobacter vinelandii ΔnifHDK Strain

The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. A His-tagged version of the NifEN complex has been previously purified and shown to carry two identical [4Fe-4S] clusters of unknown function and a [Fe-S]-containing FeMo-co precursor. We have improved the purification of the his-NifEN protein from a DeltanifHDK strain of Azotobacter vinelandii and have found that the amounts of iron and molybdenum within NifEN were significantly higher than those reported previously. In an in vitro FeMo-co synthesis system with purified components, the NifEN protein served as a source of both molybdenum and a [Fe-S]-containing FeMo-co precursor, showing significant FeMo-co synthesis activity in the absence of externally added molybdate. Thus, the NifEN scaffold protein, purified from DeltanifHDK background, contained the Nif-Bco-derived Fe-S cluster and molybdenum, although these FeMo-co constituents were present at different levels within the protein complex.

Structural insights into a protein-bound iron-molybdenum cofactor precursor

The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein is a highly complex metallocluster that provides the catalytically essential site for biological nitrogen fixation. FeMoco is assembled outside the MoFe protein in a stepwise process requiring several components, including NifB-co, an iron- and sulfur-containing FeMoco precursor, and NifEN, an intermediary assembly protein on which NifB-co is presumably converted to FeMoco. Through the comparison of Azotobacter vinelandii strains expressing the NifEN protein in the presence or absence of the nifB gene, the structure of a NifEN-bound FeMoco precursor has been analyzed by x-ray absorption spectroscopy. The results provide physical evidence to support a mechanism for FeMoco biosynthesis. The NifEN-bound precursor is found to be a molybdenum-free analog of FeMoco and not one of the more commonly suggested cluster types based on a standard [4Fe-4S] architecture. A facile scheme by which FeMoco and alternative, non-molybdenum-containing nitrogenase cofactors are constructed from this common precursor is presented that has important implications for the biosynthesis and biomimetic chemical synthesis of FeMoco.

Synthesis and characterization of homochiral polymeric S-malato molybdate(VI): toward the potentially stereospecific formation and absolute configuration of iron-molybdenum cofactor in nitrogenase

Reaction of sodium or potassium molybdate and excess malic acid in a wide range of pH values (pH 4.0–7.0) resulted in the isolation of two cis-dioxo-bis(malato)-Mo(VI) complexes, viz. Na 3[MoO 2H( S-mal) 2] and K 3[MoO 2H( S-mal) 2]·H 2O (H 3mal=malic acid). The sodium complex is also characterized by an X-ray structure analysis, showing that the mononuclear Mo units are linked together via very strong symmetric CO 2···H··· O 2C-hydrogen bond [2.432(5) Å], forming a polymeric chain. The molybdenum atoms are quasi-octahedrally coordinated by two cis-oxo groups and two bidentate malate ligands via its alkoxy and α-carboxyl groups, while the β-carboxylic and carboxylate groups remain uncomplexed, as the coordination of vicinal carboxylate and alkoxide of homocitrate in FeMo cofactor of nitrogenase. The absolute configuration of the metal center in this S-malato complex is assigned as Λ and the homochirality within the chain is established as a homochiral form ···Λ S–Λ S–Λ S–Λ S···. It is proposed that the chiral configuration of the metal center in wild-type FeMo-co biosynthesis might be induced by the early coordination of the chiral R-homocitric acid, while a mixture of raceme might be obtained in the biosynthesis of NifV − FeMo-cofactor. The absolute configuration of wild-type FeMo-cofactor is assigned as Δ R.

Tungsten–malate interaction. Synthesis, spectroscopic and structural studies of homochiral S-malato tungstate(VI), Λ-Na 3[WO 2H( S-mal) 2

Reaction of the sodium tungstate and malic acid at 1 to 2 ratio in weak acidic solution (pH 3.0–6.0) resulted in the first isolation and structural characterization of a tungsten(VI) malato complex Λ-Na 3[WO 2H( S-mal) 2] ( 1) (malic acid=H 3mal). The complex has been characterized by elemental analysis, UV and IR spectroscopy. The IR spectra are consistent with an oxo-coordinated mononuclear structure as revealed by single crystal X-ray diffraction study. The tungsten atoms are quasi-octahedrally coordinated by two cis-oxo groups and two bidentate malate ligands via its alkoxy and α-carboxyl groups, while the β-carboxylic and carboxylate groups remain uncomplexed. The latter are bonded each other through strong hydrogen bonding, forming a catenarian chain, and the anions are connected in homochiral form. It is proposed that the chiral configuration of metal center in wild-type FeMo-co biosynthesis might be induced by the coordination of the chiral R-homocitric acid, while a mixture of raceme might be obtained in the biosynthesis of NifV–FeMo-cofactor.

Syntheses and spectroscopic and structural characterization of molybdenum(VI) citrato monomeric raceme and dimer, K4[MoO3(cit)].2H2O and K4[(MoO2)2O(Hcit)2].4H2O.

Investigation of the aqueous coordination chemistry for citrate and molybdenum(VI) resulted in the isolation of molybdenum(VI) citrato monomeric raceme and dimer K4[MoO3(cit)].2H2O (1) and K4[(MoO2)2O(Hcit)2].4H2O (2) (H4cit = citric acid). Complex 1 can serve as the first structurally characterized monomeric citrato molybdate and may represent an early mobilized precursor in the biosynthesis of FeMo-co (FeMo-cofactor). The two complexes have been characterized by elemental analyses and IR and NMR spectroscopies. The IR and NMR spectra are consistent with a monomeric species or a monooxo-bridged dinuclear structure, as revealed by a single crystal X-ray diffraction study. Compound 1 is monoclinic space group P2(1)/c with a = 7.225(1) A, b = 9.151(2) A, c = 22.727(2) A, beta = 94.93(1) degrees, V = 1497.1(7) A3, and Z = 4. Full-matrix least-squares refinement resulted in residuals of R = 0.027 and Rw = 0.032. The molybdenum atom forms an octahedral coordination with three oxo groups and one tridentate citrate, in which the latter is coordinated through the alkoxy and vicinal carboxyl and much more weakly by one of the two terminal groups [2.411(3) A]. Compound 2 is triclinic space group P1 with a = 8.2728(8) A, b = 8.9514(8) A, c = 10.0605(9) A, alpha = 101.673(8) degrees, beta = 100.672(7) degrees, gamma = 112.938(7) degrees, V = 642.5(3) A3, and Z = 1. Full-matrix least-squares refinement resulted in residuals of R = 0.033 and Rw = 0.039. The complex anion contains a linear (O2Mo)O(MoO2) core with the bridging oxo group lying at the center of inversion symmetry (Mo-Ob-Mo, 180 degrees). Each citrate ligand is three-coordinated to one molybdenum atom through the deprotonated hydroxy, alpha-carboxyl, and one beta-carboxyl group, making each metal atom six-coordinate.

Inhibition of Iron-Molybdenum Cofactor Biosynthesis by L127Δ NifH and Evidence for a Complex Formation between L127Δ NifH and NifNE

Besides serving as the obligate electron donor to dinitrogenase during nitrogenase turnover, dinitrogenase reductase (NifH) is required for the biosynthesis of the iron-molybdenum cofactor (FeMo-co) and for the maturation of alpha(2)beta(2) apo-dinitrogenase (apo-dinitrogenase maturation). In an attempt to understand the role of NifH in FeMo-co biosynthesis, a site-specific altered form of NifH in which leucine at position 127 has been deleted, L127Delta, was employed in in vitro FeMo-co synthesis assays. This altered form of NifH has been shown to inhibit substrate reduction by the wild-type nitrogenase complex, forming a tight protein complex with dinitrogenase. The L127Delta NifH was found to inhibit in vitro FeMo-co synthesis by wild-type NifH as detected by the gamma gel shift assay. Increasing the concentration of NifNE and NifB-cofactor (NifB-co) relieved the inhibition of FeMo-co synthesis by L127Delta NifH. The formation of a complex of L127Delta NifH with NifNE was investigated by gel filtration chromatography. We herein report the formation of a complex between L127Delta NifH and NifNE in the presence of NifB-co. This work presents evidence for one of the possible roles for NifH in FeMo-co biosynthesis, i.e. the interaction of NifH with a NifNE.NifB-co complex.

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase: EFFECT OF SUBSTITUTION FOR NifH WITH SITE-SPECIFICALLY ALTERED FORMS OF NifH

NifH has three different roles in the nitrogenase enzyme system. Apart from serving as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (apodinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required for the aforementioned processes have been investigated by the use of site-specifically altered forms of the enzyme. The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127Delta, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH to bind and not hydrolyze MgATP is required for it to function in these processes. We also present evidence that the ability of NifH to function in these processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase. Evidence toward the existence of separate, overlapping sites on NifH for each of its functions (substrate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is presented.

In Vitro Synthesis of the Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase: EFFECT OF SUBSTITUTION OF VNFH FOR NIFH

NIFH (the nifH gene product) has several functions in the nitrogenase enzyme system. In addition to reducing dinitrogenase during nitrogenase turnover, NIFH functions in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), and in the processing of alpha2beta2 apodinitrogenase 1 (a catalytically inactive form of dinitrogenase 1 that lacks the FeMo-co) to the FeMo-co-activatable alpha2beta2gamma2 form. The molybdenum-independent nitrogenase 2 (vnf-encoded) has a distinct dinitrogenase reductase protein, VNFH. We investigated the ability of VNFH to function in the in vitro biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. VNFH can replace NIFH in both the biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. These results suggest that the dinitrogenase reductase proteins do not specify the heterometal incorporated into the cofactors of the respective nitrogenase enzymes. The specificity for the incorporation of molybdenum into FeMo-co was also examined using the in vitro FeMo-co synthesis assay system.

The Requirement of Reductant for in Vitro Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

A source of reductant is routinely added to the in vitro iron-molybdenum cofactor (FeMo-co) synthesis assay, although a requirement for reductant has not been established. This report demonstrates that the addition of reductant to the in vitro FeMo-co synthesis system is not required when Azotobacter vinelandii cell-free extract is prepared in buffer that lacks added reductant. The addition of reductant is required, however, if the A. vinelandii cell-free extract is chemically oxidized prior to addition to the assay. These results might suggest that extracts of A. vinelandii contain a physiological source of reductant that functions in the in vitro synthesis of FeMo-co. It is possible that the proteins required for FeMo-co biosynthesis (e.g. NIFNE and dinitrogenase reductase) are at the appropriate redox state to function in the in vitro reaction in the extract that is free of added reductant but not in the chemically oxidized extract. It is also possible that dinitrogenase reductase and/or NIFNE (both Fe-S proteins required for FeMo-co synthesis) might catalyze the reductant-dependent reaction for FeMo-co synthesis. Dithionite, Ti(III) citrate, and NADH are able to serve as the source of reductant for in vitro FeMo-co biosynthesis.

Biosynthesis of the iron-molybdenum cofactor of nitrogenase.

The iron-molybdenum cofactor (FeMo-co) of nitrogenase is a unique molybdenum-containing prosthetic group that has been proposed to form an integral part of the active site of dinitrogenase. In Klebsiella pneumoniae, at least six nif (nitrogen fixation) gene products are required for the biosynthesis of FeMo-co, including NIFB, NIFNE, NIFH, NIFQ, and NIFV. An in vitro system for the synthesis of FeMo-co, which requires MgATP, molybdate, homocitrate, and at least the products of nifN, E, B, and H, has provided an enzymatic assay for the purification of many of the gene products required for FeMo-co biosynthesis. Although the structure of the cofactor has been solved recently, much about the biosynthetic pathway remains unknown. This article reviews what is known about the various components required for FeMo-co biosynthesis.

Identification of the V factor needed for synthesis of the iron-molybdenum cofactor of nitrogenase as homocitrate.

Nitrogenase catalyses the ATP-dependent reduction of N2 to NH3, and is composed of two proteins, dinitrogenase (MoFe protein or component I) and dinitrogenase reductase (Fe protein or component II). Dinitrogenase contains a unique prosthetic group (iron-molybdenum cofactor, FeMoco) comprised of Fe, Mo and S, which has been proposed as the site of N2 reduction. Biochemical and genetic studies of Nif- (nitrogen fixation) mutants of Klebsiella pneumoniae which are defective in nitrogen fixation, have shown that the nifB, nifQ, nifN, nifE and nifV genes are required for the biosynthesis of FeMo-co. Recently, a system for in vitro synthesis of FeMoco was described. The assay requires at least the nifB, nifN and nifE gene products, and a low-molecular-weight factor (V factor) produced in the presence of the nifV gene product. We have used this system to study FeMoco biosynthesis. We report here the isolation of V factor and identify it as homocitric acid ([R]2-hydroxy-1,2,4-butanetricarboxylic acid).

Biosynthesis of iron-molybdenum cofactor in the absence of nitrogenase.

Klebsiella pneumoniae accumulates molybdenum during nitrogenase derepression. The molybdenum is primarily in nitrogenase component I in the form of iron-molybdenum cofactor (FeMo-co). Mutations in any of three genes (nifB, nifN, and nifE) involved in the biosynthesis of FeMo-co resulted in very low molybdenum accumulation and in a molybdenum-free nitrogenase component I. A mutant lacking both subunits of nitrogenase component I accumulated 60% of the amount of molybdenum present in the wild type. The molybdenum was in protein-bound form and behaved differently than that in the wild type with respect to electrophoretic mobility, size, and extractability by organic solvents. Two forms of molybdenum could be extracted from the protein fraction of the mutant; one of them was not detected in the wild type, and the other behaved like FeMo-co in nonaqueous gel filtration chromatography. Crude extracts of this mutant were able to complement in vitro K. pneumoniae or Azotobacter vinelandii mutants unable to produce FeMo-co. These data show that biosynthesis of FeMo-co does not require the presence of nitrogenase component I. In its absence, FeMo-co is accumulated on a different protein, presumably an intermediate in the normal FeMo-co biosynthetic pathway.