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Nonenzymatic synthesis of the P-cluster in the nitrogenase MoFe protein: evidence of the involvement of all-ferrous [Fe4S4](0) intermediates.

ABSTRACT: The P-cluster in the nitrogenase MoFe protein is a [Fe8S7] cluster and represents the most complex FeS cluster found in Nature. To date, the exact mechanism of the in vivo synthesis of the P-cluster remains unclear. What is known is that the precursor to the P-cluster is a pair of neighboring [Fe4S4]-like clusters found on the ΔnifH MoFe protein, a protein expressed in the absence of the nitrogenase Fe protein (NifH). Moreover, incubation of the ΔnifH MoFe protein with NifH and MgATP results in the synthesis of the MoFe protein P-clusters. To improve our understanding of the mechanism of this reaction, we conducted a magnetic circular dichroism (MCD) spectroscopic study of the [Fe4S4]-like clusters on the ΔnifH MoFe protein. Reducing the ΔnifH MoFe protein with Ti(III) citrate results in the quenching of the S = (1)/2 electron paramagnetic resonance signal associated with the [Fe4S4](+) state of the clusters. MCD spectroscopy reveals this reduction results in all four 4Fe clusters being converted into the unusual, all-ferrous [Fe4S4](0) state. Subsequent increases of the redox potential generate new clusters. Most significantly, one of these newly formed clusters is the P-cluster, which represents approximately 20-25% of the converted Fe concentration. The other two clusters are an X cluster, of unknown structure, and a classic [Fe4S4] cluster, which represents approximately 30-35% of the Fe concentration. Diamagnetic FeS clusters may also have been generated but, because of their low spectral intensity, would not have been identified. These results demonstrate that the nitrogenase P-cluster can be generated in the absence of NifH and MgATP.

SUBMITTER: Rupnik K

PROVIDER: S-EPMC3970913 | biostudies-literature |

REPOSITORIES: biostudies-literature

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P-cluster maturation on nitrogenase MoFe protein.

Project description:Biosynthesis of nitrogenase P-cluster has attracted considerable attention because it is biologically important and chemically unprecedented. Previous studies suggest that P-cluster is formed from a precursor consisting of paired [4Fe-4S]-like clusters and that P-cluster is assembled stepwise on MoFe protein, i.e., one cluster is assembled before the other. Here, we specifically tackle the assembly of the second P-cluster by combined biochemical and spectroscopic approaches. By using a P-cluster maturation assay that is based on purified components, we show that the maturation of the second P-cluster requires the concerted action of NifZ, Fe protein, and MgATP and that the action of NifZ is required before that of Fe protein/MgATP, suggesting that NifZ may act as a chaperone that facilitates the subsequent action of Fe protein/MgATP. Furthermore, we provide spectroscopic evidence that the [4Fe-4S] cluster-like fragments can be converted to P-clusters, thereby firmly establishing the physiological relevance of the previously identified P-cluster precursor.

| S-EPMC1965529 | biostudies-literature

P+ state of nitrogenase p-cluster exhibits electronic structure of a [Fe4S4]+ cluster.

Project description:Mo nitrogenase consists of two component proteins: the Fe protein, which contains a [Fe(4)S(4)] cluster, and the MoFe protein, which contains two different classes of metal cluster: P-cluster ([Fe(8)S(7)]) and FeMoco ([MoFe(7)S(9)C·homocitrate]). The P-cluster is believed to mediate the electron transfer between the Fe protein and the MoFe protein via interconversions between its various oxidation states, such as the all-ferrous state (P(N)) and the one- (P(+)) and two-electron (P(2+)) oxidized states. While the structural and electronic properties of P(N) and P(2+) states have been well characterized, little is known about the electronic structure of the P(+) state. Here, a mutant strain of Azotobacter vinelandii (DJ1193) was used to facilitate the characterization of the P(+) state of P-cluster. This strain expresses a MoFe protein variant (designated ?nifB ?-188(Cys) MoFe protein) that accumulates the P(+) form of P-cluster in the resting state. Magnetic circular dichroism (MCD) spectrum of the P-cluster in the oxidized ?nifB ?-188(Cys) MoFe protein closely resembles that of the P(2+) state in the oxidized wild-type MoFe protein, except for the absence of a major charge-transfer band centered at 823 nm. Moreover, magnetization curves of ?nifB ?-188(Cys) and wild-type MoFe proteins suggest that the P(2+) species in both proteins have the same spin state. MCD spectrum of the P(+) state in the ?nifB ?-188(Cys) MoFe protein, on the other hand, is associated with a classic [Fe(4)S(4)](+) cluster, suggesting that the P-cluster could be viewed as two coupled 4Fe clusters and that it could donate either one or two electrons to FeMoco by using one or both of its 4Fe halves. Such a mode of action of P-cluster could provide energetic and kinetic advantages to nitrogenase in the complex mechanism of N(2) reduction.

| S-EPMC3442605 | biostudies-literature

Dissecting Electronic-Structural Transitions in the Nitrogenase MoFe Protein P-Cluster during Reduction.

Project description:The [8Fe-7S] P-cluster of nitrogenase MoFe protein mediates electron transfer from nitrogenase Fe protein during the catalytic production of ammonia. The P-cluster transitions between three oxidation states, PN, P+, P2+ of which PN↔P+ is critical to electron exchange in the nitrogenase complex during turnover. To dissect the steps in formation of P+ during electron transfer, photochemical reduction of MoFe protein at 231-263 K was used to trap formation of P+ intermediates for analysis by EPR. In complexes with CdS nanocrystals, illumination of MoFe protein led to reduction of the P-cluster P2+ that was coincident with formation of three distinct EPR signals: S = 1/2 axial and rhombic signals, and a high-spin S = 7/2 signal. Under dark annealing the axial and high-spin signal intensities declined, which coincided with an increase in the rhombic signal intensity. A fit of the time-dependent changes of the axial and high-spin signals to a reaction model demonstrates they are intermediates in the formation of the P-cluster P+ resting state and defines how spin-state transitions are coupled to changes in P-cluster oxidation state in MoFe protein during electron transfer.

| S-EPMC8991001 | biostudies-literature

Chimeric Interaction of Nitrogenase-Like Reductases with the MoFe Protein of Nitrogenase.

Project description:The engineering of transgenic organisms with the ability to fix nitrogen is an attractive possibility. However, oxygen sensitivity of nitrogenase, mainly conferred by the reductase component (NifH)2 , is an imminent problem. Nitrogenase-like enzymes involved in coenzyme F430 and chlorophyll biosynthesis utilize the highly homologous reductases (CfbC)2 and (ChlL)2 , respectively. Chimeric protein-protein interactions of these reductases with the catalytic component of nitrogenase (MoFe protein) did not support nitrogenase activity. Nucleotide-dependent association and dissociation of these complexes was investigated, but (CfbC)2 and wild-type (ChlL)2 showed no modulation of the binding affinity. By contrast, the interaction between the (ChlL)2 mutant Y127S and the MoFe protein was markedly increased in the presence of ATP (or ATP analogues) and reduced in the ADP state. Upon formation of the octameric (ChlL)2 MoFe(ChlL)2 complex, the ATPase activity of this variant is triggered, as seen in the homologous nitrogenase system. Thus, the described reductase(s) might be an attractive tool for further elucidation of the diverse functions of (NifH)2 and the rational design of a more robust reductase.

| S-EPMC7317204 | biostudies-literature

Molybdenum L-Edge XAS Spectra of MoFe Nitrogenase.

Project description:A molybdenum L-edge X-ray absorption spectroscopy (XAS) study is presented for native and oxidized MoFe protein of nitrogenase as well as Mo-Fe model compounds. Recently collected data on MoFe protein (in oxidized and reduced forms) is compared to previously published Mo XAS data on the isolated FeMo cofactor in NMF solution and put in context of the recent Mo K-edge XAS study, which showed a MoIII assignment for the molybdenum atom in FeMoco. The L3-edge data are interpreted within a simple ligand-field model, from which a time-dependent density functional theory (TDDFT) approach is proposed as a way to provide further insights into the analysis of the molybdenum L3-edges. The calculated results reproduce well the relative spectral trends that are observed experimentally. Ultimately, these results give further support for the MoIII assignment in protein-bound FeMoco, as well as isolated FeMoco.

| S-EPMC4510703 | biostudies-literature

The sixteenth iron in the nitrogenase MoFe protein.

Project description:Another iron in the fire: X-ray anomalous diffraction studies on the nitrogenase MoFe protein show the presence of a mononuclear iron site, designated as Fe16, which was previously identified as either Ca(2+) or Mg(2+). The position of the absorption edge indicates that this site is in the oxidation state +2. The high sequence conservation of the residues coordinated to Fe16 emphasizes the potential importance of the site in nitrogenase.

| S-EPMC3891402 | biostudies-literature

A substrate channel in the nitrogenase MoFe protein.

Project description:Nitrogenase catalyzes the six electron/six proton reduction of N2 to two ammonia molecules at a complex organometallocluster called "FeMo cofactor." This cofactor is buried within the alpha-subunit of the MoFe protein, with no obvious access for substrates. Examination of high-resolution X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent-exposed surface to a specific face of FeMo cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate reduction kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters were established for the reduction of the substrates N2, acetylene, azide, and propyne. For each MoFe protein, V (max) values for the different substrates were found to be nearly unchanged when compared with the values for the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, the K(m) values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared with the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water-filled channel functioning as a substrate channel in the MoFe protein.

| S-EPMC2819195 | biostudies-literature

Protein dynamics and the all-ferrous [Fe4 S4 ] cluster in the nitrogenase iron protein.

Project description:In nitrogen fixation by Azotobacter vinelandii nitrogenase, the iron protein (FeP) binds to and subsequently transfers electrons to the molybdenum-FeP, which contains the nitrogen fixation site, along with hydrolysis of two ATPs. However, the nature of the reduced state cluster is not completely clear. While reduced FeP is generally thought to contain an [Fe4 S4 ](1+) cluster, evidence also exists for an all-ferrous [Fe4 S4 ](0) cluster. Since the former indicates a single electron is transferred per two ATPs hydrolyzed while the latter indicates two electrons could be transferred per two ATPs hydrolyzed, an all-ferrous [Fe4 S4 ](0) cluster in FeP is potenially two times more efficient. However, the 1+/0 reduction potential has been measured in the protein at both 460 and 790 mV, causing the biological significance to be questioned. Here, "density functional theory plus Poisson Boltzmann" calculations show that cluster movement relative to the protein surface observed in the crystal structures could account for both measured values. In addition, elastic network mode analysis indicates that such movement occurs in low frequency vibrations of the protein, implying protein dynamics might lead to variations in reduction potential. Furthermore, the different reductants used in the conflicting measurements of the reduction potential could be differentially affecting the protein dynamics. Moreover, even if the all-ferrous cluster is not the biologically relevant cluster, mutagenesis to stabilize the conformation with the more exposed cluster may be useful for bioengineering more efficient enzymes.

| S-EPMC4815322 | biostudies-literature

Nitrogenase MoFe protein from Clostridium pasteurianum at 1.08 A resolution: comparison with the Azotobacter vinelandii MoFe protein.

Project description:The X-ray crystal structure of the nitrogenase MoFe protein from Clostridium pasteurianum (Cp1) has been determined at 1.08 Å resolution by multiwavelength anomalous diffraction phasing. Cp1 and the ortholog from Azotobacter vinelandii (Av1) represent two distinct families of nitrogenases, differing primarily by a long insertion in the α-subunit and a deletion in the β-subunit of Cp1 relative to Av1. Comparison of these two MoFe protein structures at atomic resolution reveals conserved structural arrangements that are significant to the function of nitrogenase. The FeMo cofactors defining the active sites of the MoFe protein are essentially identical between the two proteins. The surrounding environment is also highly conserved, suggesting that this structural arrangement is crucial for nitrogen reduction. The P clusters are likewise similar, although the surrounding protein and solvent environment is less conserved relative to that of the FeMo cofactor. The P cluster and FeMo cofactor in Av1 and Cp1 are connected through a conserved water tunnel surrounded by similar secondary-structure elements. The long α-subunit insertion loop occludes the presumed Fe protein docking surface on Cp1 with few contacts to the remainder of the protein. This makes it plausible that this loop is repositioned to open up the Fe protein docking surface for complex formation.

| S-EPMC4321486 | biostudies-literature

Structural Analysis of a Nitrogenase Iron Protein from Methanosarcina acetivorans: Implications for CO2 Capture by a Surface-Exposed [Fe4S4] Cluster.

Project description:Nitrogenase iron (Fe) proteins reduce CO2 to CO and/or hydrocarbons under ambient conditions. Here, we report a 2.4-Å crystal structure of the Fe protein from Methanosarcina acetivorans (MaNifH), which is generated in the presence of a reductant, dithionite, and an alternative CO2 source, bicarbonate. Structural analysis of this methanogen Fe protein species suggests that CO2 is possibly captured in an unactivated, linear conformation near the [Fe4S4] cluster of MaNifH by a conserved arginine (Arg) pair in a concerted and, possibly, asymmetric manner. Density functional theory calculations and mutational analyses provide further support for the capture of CO2 on MaNifH while suggesting a possible role of Arg in the initial coordination of CO2 via hydrogen bonding and electrostatic interactions. These results provide a useful framework for further mechanistic investigations of CO2 activation by a surface-exposed [Fe4S4] cluster, which may facilitate future development of FeS catalysts for ambient conversion of CO2 into valuable chemical commodities.IMPORTANCE This work reports the crystal structure of a previously uncharacterized Fe protein from a methanogenic organism, which provides important insights into the structural properties of the less-characterized, yet highly interesting archaeal nitrogenase enzymes. Moreover, the structure-derived implications for CO2 capture by a surface-exposed [Fe4S4] cluster point to the possibility of developing novel strategies for CO2 sequestration while providing the initial insights into the unique mechanism of FeS-based CO2 activation.

| S-EPMC6747716 | biostudies-literature

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