Information

E5. Oxidation States of Nitrogenase Fe centers - Biology


First Half: It would be difficult to assign specific oxidation states to each Fe ion in the M complex. We will first assign this to a average Fe ion, M0, with an arbitrarily assigned oxidation state of 0. On addition of 1 electron, the oxidation state would go from M0 to M-1 as the metal is reduced. The M-1 state is then oxidized as an electron is transferred to H+, and when 2 electrons are transferred, and a single H- is made. The diagram below shows the change in oxidation state in going from E0 to E4.

The red boxes highlight thermodynamic cycle-like steps which show how the changes in redox state of the Fe ions (M) could be visualized. Note that in going from E0 to E4, the actual oxidation state of M changes from 0 to +1 to 0 to +1 and back to 0. That is quite amazing given that 4 electrons have been added. Note also that in the red box going from step E0 to E1, M goes from -1 to +1 which corresponds to our description of an oxidative addition when the metal center looses two electrons. This mechanism shows that nitrogenase could be considered a "hydride storage device".

Second Half (facing forward to production NH3):

How does N2 initially interact with E4? It must depend on how the hydrides are released as H2, which evidence shows occurs by reductive elimination (re) and not hydride protonation (hp). On addition, the N2 very quickly is converted to diazene, HN=NH, with the departing H2 taking with it 2 H+s and 2 electrons (or reducing equivalents). These events could occur as shown in the figure below.

Now, with N2 bound as diazene (N2H2) and H2 released, the rest of the reaction could occur as shown below. One new step, a migratory insertion, is shown.

The two halves of the reaction are similar with bridging hydrides utilized. The E4 Janus intermediate links the two halves together.

Contributors

  • Prof. Henry Jakubowski (College of St. Benedict/St. John's University)

E5. Oxidation States of Nitrogenase Fe centers - Biology

a Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36, 45470 Mülheim an der Ruhr, Germany
E-mail: [email protected]
Tel: +49 208 306 3605

b Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700, USA
E-mail: [email protected]

c Science Institute, University of Iceland, Dunhagi 3, 107 Reykjavik, Iceland

d Centro Nacional de Pesquisa em Energia e Materiais Brazilian Synchrotron Light Laboratory - LNLS Rua Giuseppe Máximo Scolfaro, 10.000 13083-970 Campinas SP, Brazil

e Institute for Biochemistry and BIOSS Centre for Biological Signalling Studies, Albert Ludwigs University Freiburg, Germany
E-mail: [email protected]

f Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA

Abstract

An investigation of the active site cofactors of the molybdenum and vanadium nitrogenases (FeMoco and FeVco) was performed using high-resolution X-ray spectroscopy. Synthetic heterometallic iron–sulfur cluster models and density functional theory calculations complement the study of the MoFe and VFe holoproteins using both non-resonant and resonant X-ray emission spectroscopy. Spectroscopic data show the presence of direct iron–heterometal bonds, which are found to be weaker in FeVco. Furthermore, the interstitial carbide is found to perturb the electronic structures of the cofactors through highly covalent Fe–C bonding. The implications of these conclusions are discussed in light of the differential reactivity of the molybdenum and vanadium nitrogenases towards various substrates. Possible functional roles for both the heterometal and the interstitial carbide are detailed.


Introduction

Biological nitrogen fixation is an essential process required to tap into the predominant reservoir of bioavailable nitrogen, atmospheric N2. Although at any given time an estimated 99% of all nitrogen cycling the biosphere are present in this form 1 , only a single enzymatic reaction has evolved to break the stable triple bond of the dinitrogen molecule. The enzyme nitrogenase is a complex two-component system consisting of the heterotetrameric MoFe protein (NifD2K2), where substrates are reduced, and the dimeric Fe protein (NifH2) that serves as the only known electron donor for N2 reduction and as the site of ATP hydrolysis 2,3 . Nitrogenase reduces N2 according to equation (1), as well as a series of alternative substrates, prominently including carbon monoxide that is converted to a mixture of unsaturated hydrocarbons with potential relevance for biofuel production 4,5 .

The MoFe protein of nitrogenase contains two unique metal clusters, the [8Fe:7S] P-cluster, an electron transfer centre with an unusual 8Fe +2 ground state as isolated, and FeMo cofactor (FeMoco), the site of substrate reduction. FeMoco was originally described as a [Mo:7Fe:9S]:R-homocitrate entity with a central cavity surrounded by six coordinatively unsaturated iron ions 6,7 . The arrangement intuitively suggested a coordination site for substrates within the central cavity, but this hypothesis was challenged by the structural inertness of the cluster. We then found that a central light atom that was masked by an unfavourable occurrence of Fourier series termination artefacts was eventually located in the cofactor centre 8 . Only recently this light atom was identified as a carbon species 9,10 that originates from S-adenosylmethionine 11 and does not exchange during catalysis 12 . It thus constitutes a stabilizing element that provides rigidity to the cluster ground state and explains the observed structural homogeneity. Nitrogenase MoFe protein is commonly isolated in a reduced form, with a diamagnetic, all-ferrous P-cluster (P N ) and the FeMo cofactor in an S=3/2 state (FeMoco N ) that has been extensively characterized by electron paramagnetic resonance (EPR) and enhanced nuclear double-resonance (ENDOR), X-ray absorption spectroscopy (XAS) and Mössbauer spectroscopies 13,14,15,16,17 . A recent analysis of single-crystal EPR data for the FeMoco N state revealed the orientation of the magnetic g-tensor of the cluster within MoFe protein, highlighting that the electrostatic potential field induced by the protein matrix orients the spin system 18 . In this study, the gz main axis of the magnetic tensor oriented along the intrinsic threefold symmetry axis of FeMo cofactor, whereas the gy axis was found to align with a cluster edge formed by atoms Fe1, Fe3, Fe7 and Mo (Fig. 1a).

(a) Structure of FeMo cofactor as observed in MoFe protein from A. vinelandii (PDB-ID 3U7Q). The structure represents the S=3/2 FeMoco N state, a stable resting state that does not bind substrates. (b) Strategy for spatially resolved anomalous dispersion (SpReAD) refinement. A XAS spectrum or a fluorescence scan of the iron K-edge (red) is chosen to determine energies for the collection of full diffraction data sets. In these, the anomalous scattering contribution can be refined for individual atoms. (c) Anomalous difference electron density maps contoured at the 3σ level around FeMo cofactor, calculated for data sets taken at the indicated positions along the iron K-edge. The magnitude of the electron density peak does not directly reflect f″, but the increase of signal is clearly visible. Note that strong features appear first for the most electron-rich atoms, Fe1, Fe3 and Fe7 (orientation as in a).

The final clarification of the structure of FeMo cofactor, however, did not help to answer key mechanistic question, and the exact binding site for substrates as well as the mechanism of their reduction remain to be elucidated (Table 1). A deeper understanding of complex bioinorganic systems commonly arises only from a combination of methodologies, with structural information complemented by spectroscopy, but also by theoretical calculations and synthetic models. Small-molecule compounds able to activate dinitrogen were known for decades 19 , but only more recent compounds, such as [HIPTN3N]Mo(N2) by Schrock 20 and [Mo(N2)2(PNP)]2(μ-N2) by Nishibayashi 21 , achieved several cycles of catalytic turnover and were based on molybdenum as the reactive species. More recently, Hou and co-workers presented a catalytic titanium hydride complex, [(C5Me4SiMe3)Ti]4(μ 3 -NH)2(μ 2 -H)4 22 . Peters et al. synthesized a first iron compound [(TBP)Fe(N2)] that achieved catalysis 23 , and Holland and co-workers most recently extended this to a Fe-based system with iron and carbon ligands that is chemically reminiscent of FeMoco 24 . Each of these studies represented a major advance in synthetic inorganic chemistry and the catalysis of dinitrogen reduction, but they did not provide unambiguous evidence for the mode of action of the enzyme. Because of the inertness of the resting state, the investigation of inhibitor/substrate interactions with FeMo cofactor was limited to spectroscopic methods, and here an incomplete understanding of the electronic structure of FeMoco precluded the precise assignment of signals 19 . Very recently, the discovery of CO-bound FeMoco by X-ray crystallography provided structural information on a functionalized state of the active site for the first time 25 . The utility of such structural data for guiding further spectroscopic and theoretical studies is obvious and may help to advance understanding of the mechanism of nitrogenase substantially. However, a prerequisite for this approach is a detailed electronic description of FeMoco, and in spite of numerous studies by many of the leaders is their field, theoretical approaches to date have not succeeded to produce a generally accepted model 19 . Different models proposed ligand binding to the central cavity 26,27 , but also to various positions on the cluster surface 28,29 , and most recently the CO adduct inspired a proposal where N2 is activated in the exact same position, made possible through concomitant H2 evolution 30 .

Experimental data on the actual electron distribution within FeMoco are scarce, largely because spectroscopic techniques can hardly resolve and characterize individual Fe ions in among the 30 Fe sites per MoFe protein. On the other hand, the precise spatial resolution of experimental data is a hallmark of diffraction techniques, but although X-ray crystallography was highly instrumental in describing the structure of the cluster, it is not straightforward to use as a tool for investigating oxidation states of individual metals. We have recently presented a method to address this problem and extract further information about the electronic structure of every single metal site from X-ray diffraction data 31 . This strategy exploits the property of anomalous scattering that describes the breakdown of Friedel’s law 32 , the intrinsic inversion symmetry of diffraction (F(S)=F(–S)), in the proximity of an X-ray absorption edge. Anomalous scattering is routinely used to solve the crystallographic phase problem for the ab initio structure determination of proteins 33 , and its magnitude across an edge is proportional to the absorption of X-rays. With diffraction data sets collected at various X-ray energies, it is possible to refine the anomalous scattering contributions f ″ and f ′ individually for each anomalous scatterer in a structure (Fig. 1b and Supplementary Table 1). The relevance of such a spatially resolved anomalous dispersion (SpReAD) analysis arises from the fact that the absorption properties of a given scatterer reflect the chemical environment, but also the electronic state of the atom. Upon oxidation, the ionization energy of the inner-shell electrons that determines the position of an absorption edge will increase, making the edge position a strong indicator for oxidation state and enabling us to carry out this analysis at any given point in space, that is, for each single atom of a given type. The method was successfully applied to identify the localized electron in a reduced [2Fe:2S] cluster 31 , and to characterize an additional Fe site recently discovered in MoFe protein 34 .

Here we have applied the SpReAD methodology to the enzyme nitrogenase as one of the largest and most complex metalloproteins known to date. We show that the edge positions, and thus most likely the oxidation states of the individual iron sites are distinct and suggest an electron distribution that matches up with the Mo(III) ion present in the cluster to yield the observed S=3/2 state. Understanding the electronic structure of FeMoco is an essential prerequisite for a concise functional analysis of this unique centre, paving the way for possible applications in catalysis and bioengineering.


Contents

Although the equilibrium formation of ammonia from molecular hydrogen and nitrogen has an overall negative enthalpy of reaction ( Δ H 0 = − 45.2 k J m o l − 1 N H 3 =-45.2 mathrm ,mathrm > mathrm > > ), the activation energy is very high ( E A = 230 − 420 k J m o l − 1 =230-420 mathrm ,mathrm > > ). [1] Nitrogenase acts as a catalyst, reducing this energy barrier such that the reaction can take place at ambient temperatures.

A usual assembly consists of two components:

  1. The heterotetrameric MoFe protein, a nitrogenase which uses the electrons provided to reduce N2 to NH3. In some assemblies it is replaced by a homologous alternative.
  2. The homodimeric Fe-only protein, the reductase which has a high reducing power and is responsible for the supply of electrons.

Reductase Edit

The Fe protein, the dinitrogenase reductase or NifH, is a dimer of identical subunits which contains one [Fe4S4] cluster and has a mass of approximately 60-64kDa. [2] The function of the Fe protein is to transfer electrons from a reducing agent, such as ferredoxin or flavodoxin to the nitrogenase protein. The transfer of electrons requires an input of chemical energy which comes from the binding and hydrolysis of ATP. The hydrolysis of ATP also causes a conformational change within the nitrogenase complex, bringing the Fe protein and MoFe protein closer together for easier electron transfer. [3]

Nitrogenase Edit

The MoFe protein is a heterotetramer consisting of two α subunits and two β subunits, with a mass of approximately 240-250kDa. [2] The MoFe protein also contains two iron-sulfur clusters, known as P-clusters, located at the interface between the α and β subunits and two FeMo cofactors, within the α subunits. The oxidation state of Mo in these nitrogenases was formerly thought Mo(V), but more recent evidence is for Mo(III). [4] (Molybdenum in other enzymes is generally bound to molybdopterin as fully oxidized Mo(VI)).

  • The core (Fe8S7) of the P-cluster takes the form of two [Fe4S3] cubes linked by a central sulfur atom. Each P-cluster is covalently linked to the MoFe protein by six cysteine residues.
  • Each FeMo cofactor (Fe7MoS9C) consists of two non-identical clusters: [Fe4S3] and [MoFe3S3], which are linked by three sulfide ions. Each FeMo cofactor is covalently linked to the α subunit of the protein by one cysteine residue and one histidine residue.

Electrons from the Fe protein enter the MoFe protein at the P-clusters, which then transfer the electrons to the FeMo cofactors. Each FeMo cofactor then acts as a site for nitrogen fixation, with N2 binding in the central cavity of the cofactor.

Variations Edit

The MoFe protein can be replaced by alternative nitrogenases in environments low in the Mo cofactor. Two types of such nitrogenases are known: the vanadium-iron (VFe Vnf) type and the iron-iron (FeFe Anf) type. Both form an assembly of two α subunits, two β subunits, and two δ (sometimes γ) subunits. The delta subunits are homologous to each other, and the alpha and beta subunits themselves are homologous to the ones found in MoFe nitrogenase. The gene clusters are also homologous, and these subunits are interchangeable to some degree. All nitrogenases use a similar Fe-S core cluster, and the variations come in the cofactor metal. [5] [6]

The Anf nitrogenase in Azotobacter vinelandii is organized in an anfHDGKOR operon. This operon still requires some of the Nif genes to function. An engineered minimal 10-gene operon that incorporates these additional essential genes has been constructed. [7]

General mechanism Edit

Nitrogenase is an enzyme responsible for catalyzing nitrogen fixation, which is the reduction of nitrogen (N2) to ammonia (NH3) and a process vital to sustaining life on Earth. [8] There are three types of nitrogenase found in various nitrogen-fixing bacteria: molybdenum (Mo) nitrogenase, vanadium (V) nitrogenase, and iron-only (Fe) nitrogenase. [9] Molybdenum nitrogenase, which can be found in diazotrophs such as legume-associated rhizobia, [10] [11] is the nitrogenase that has been studied the most extensively and thus is the most well characterized. [9] Figures 1-2 display the crystal structure and key catalytic components of molybdenum nitrogenase extracted from Azotobacter vinelandii. Vanadium nitrogenase and iron-only nitrogenase can both be found in select species of Azotobacter as an alternative nitrogenase. [10] [12] Equations 1 and 2 show the balanced reactions of nitrogen fixation in molybdenum nitrogenase and vanadium nitrogenase respectively.

All nitrogenases are two-component systems made up of Component I (also known as dinitrogenase) and Component II (also known as dinitrogenase reductase). Component I is a MoFe protein in molybdenum nitrogenase, a VFe protein in vanadium nitrogenase, and a Fe protein in iron-only nitrogenase. [8] Component II is a Fe protein that contains the Fe-S cluster (Figure 3: top), which transfers electrons to Component I. [12] Component I contains 2 key metal clusters: the P-cluster (Figure 3: middle), and the FeMo-cofactor (FeMo-co) (Figure 3: bottom). Mo is replaced by V or Fe in vanadium nitrogenase and iron-only nitrogenase respectively. [8] During catalysis, electrons flow from a pair of ATP molecules within Component II to the Fe-S cluster, to the P-cluster, and finally to the FeMo-co, where reduction of N2 to NH3 takes place.

Lowe-Thorneley kinetic model Edit

The reduction of nitrogen to two molecules of ammonia is carried out at the FeMo-co of Component I after the sequential addition of proton and electron equivalents from Component II. [8] Steady state, freeze quench, and stopped-flow kinetics measurements carried out in the 70's and 80's by Lowe, Thorneley, and others provided a kinetic basis for this process. [14] [15] The Lowe-Thorneley (LT) kinetic model (depicted in Figure 4) was developed from these experiments and documents the eight correlated proton and electron transfers required throughout the reaction. [8] [14] [15] Each intermediate stage is depicted as En where n = 0-8, corresponding to the number of equivalents transferred. The transfer of four equivalents are required before the productive addition of N2, although reaction of E3 with N2 is also possible. [14] Notably, nitrogen reduction has been shown to require 8 equivalents of protons and electrons as opposed to the 6 equivalents predicted by the balanced chemical reaction. [16]

Intermediates E0 through E4 Edit

Spectroscopic characterization of these intermediates has allowed for greater understanding of nitrogen reduction by nitrogenase, however, the mechanism remains an active area of research and debate. Briefly listed below are spectroscopic experiments for the intermediates before the addition of nitrogen:

E0 – This is the resting state of the enzyme before catalysis begins. EPR characterization shows that this species has a spin of 3 /2. [17]

E1 – The one electron reduced intermediate has been trapped during turnover under N2. Mӧssbauer spectroscopy of the trapped intermediate indicates that the FeMo-co is integer spin greater than 1. [18]

E2 – This intermediate is proposed to contain the metal cluster in its resting oxidation state with the two added electrons stored in a bridging hydride and the additional proton bonded to a sulfur atom. Isolation of this intermediate in mutated enzymes shows that the FeMo-co is high spin and has a spin of 3 /2. [19]

E3 – This intermediate is proposed to be the singly reduced FeMo-co with one bridging hydride and one proton. [8]

E4 – Termed the Janus intermediate after the Roman god of transitions, this intermediate is positioned after exactly half of the electron proton transfers and can either decay back to E0 or proceed with nitrogen binding and finish the catalytic cycle. This intermediate is proposed to contain the FeMo-co in its resting oxidation state with two bridging hydrides and two sulfur bonded protons. [8] This intermediate was first observed using freeze quench techniques with a mutated protein in which residue 70, a valine amino acid, is replaced with isoleucine. [20] This modification prevents substrate access to the FeMo-co. EPR characterization of this isolated intermediate shows a new species with a spin of ½. ENDOR experiments have provided insight into the structure of this intermediate, revealing the presence of two bridging hydrides. [20] 95 Mo and 57 Fe ENDOR show that the hydrides bridge between two iron centers. [21] Cryoannealing of the trapped intermediate at -20 °C results in the successive loss of two hydrogen equivalents upon relaxation, proving that the isolated intermediate is consistent with the E4 state. [8] The decay of E4 to E2 + H2 and finally to E0 and 2H2 has confirmed the EPR signal associated with the E2 intermediate. [8]

The above intermediates suggest that the metal cluster is cycled between its original oxidation state and a singly reduced state with additional electrons being stored in hydrides. It has alternatively been proposed that each step involves the formation of a hydride and that the metal cluster actually cycles between the original oxidation state and a singly oxidized state. [8]

Distal and alternating pathways for N2 fixation Edit

While the mechanism for nitrogen fixation prior to the Janus E4 complex is generally agreed upon, there are currently two hypotheses for the exact pathway in the second half of the mechanism: the "distal" and the "alternating" pathway (see Figure 5). [8] [22] [23] In the distal pathway, the terminal nitrogen is hydrogenated first, releases ammonia, then the nitrogen directly bound to the metal is hydrogenated. In the alternating pathway, one hydrogen is added to the terminal nitrogen, then one hydrogen is added to the nitrogen directly bound to the metal. This alternating pattern continues until ammonia is released. [8] [22] [23] Because each pathway favors a unique set of intermediates, attempts to determine which path is correct have generally focused on the isolation of said intermediates, such as the nitrido in the distal pathway, [24] and the diazene and hydrazine in the alternating pathway. [8] Attempts to isolate the intermediates in nitrogenase itself have so far been unsuccessful, but the use of model complexes has allowed for the isolation of intermediates that support both sides depending on the metal center used. [8] Studies with Mo generally point towards a distal pathway, while studies with Fe generally point towards an alternating pathway. [8] [22] [23] [25] [26]

Specific support for the distal pathway has mainly stemmed from the work of Schrock and Chatt, who successfully isolated the nitrido complex using Mo as the metal center in a model complex. [24] [27] Specific support for the alternating pathway stems from a few studies. Iron only model clusters have been shown to catalytically reduce N2. [25] [26] Small tungsten clusters have also been shown to follow an alternating pathway for nitrogen fixation. [28] The vanadium nitrogenase releases hydrazine, an intermediate specific to the alternating mechanism. [8] [29] However, the lack of characterized intermediates in the native enzyme itself means that neither pathway has been definitively proven. Furthermore, computational studies have been found to support both sides, depending on whether the reaction site is assumed to be at Mo (distal) or at Fe (alternating) in the MoFe cofactor. [8] [22] [23]

Mechanism of MgATP binding Edit

Binding of MgATP is one of the central events to occur in the mechanism employed by nitrogenase. Hydrolysis of the terminal phosphate group of MgATP provides the energy needed to transfer electrons from the Fe protein to the MoFe protein. [30] The binding interactions between the MgATP phosphate groups and the amino acid residues of the Fe protein are well understood by comparing to similar enzymes, while the interactions with the rest of the molecule are more elusive due to the lack of a Fe protein crystal structure with MgATP bound (as of 1996). [31] Three protein residues have been shown to have significant interactions with the phosphates, shown in Figure 6. [14] In the absence of MgATP, a salt bridge exists between residue 15, lysine, and residue 125, aspartic acid. [31] Upon binding, this salt bridge is interrupted. Site-specific mutagenesis has demonstrated that when the lysine is substituted for a glutamine, the protein's affinity for MgATP is greatly reduced [32] and when the lysine is substituted for an arginine, MgATP cannot bind due to the salt bridge being too strong. [33] The necessity of specifically aspartic acid at site 125 has been shown through noting altered reactivity upon mutation of this residue to glutamic acid. [34] The third residue that has been shown to be key for MgATP binding is residue 16, serine. Site-specific mutagenesis was used to demonstrate this fact. [34] This has led to a model in which the serine remains coordinated to the Mg 2+ ion after phosphate hydrolysis in order to facilitate its association with a different phosphate of the now ADP molecule. [35] MgATP binding also induces significant conformational changes within the Fe protein. [14] Site-directed mutagenesis was employed to create mutants in which MgATP binds but does not induce a conformational change. [36] Comparing X-ray scattering data in the mutants versus in the wild-type protein led to the conclusion that the entire protein contracts upon MgATP binding, with a decrease in radius of approximately 2.0 Å. [36]

Other mechanistic details Edit

Many mechanistic aspects of catalysis remain unknown. No crystallographic analysis has been reported on substrate bound to nitrogenase.

Nitrogenase is able to reduce acetylene, but is inhibited by carbon monoxide, which binds to the enzyme and thereby prevents binding of dinitrogen. Dinitrogen prevent acetylene binding, but acetylene does not inhibit binding of dinitrogen and requires only one electron for reduction to ethylene. [37] Due to the oxidative properties of oxygen, most nitrogenases are irreversibly inhibited by dioxygen, which degradatively oxidizes the Fe-S cofactors. [ citation needed ] This requires mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo. Despite this problem, many use oxygen as a terminal electron acceptor for respiration. [ citation needed ] Although the ability of some nitrogen fixers such as Azotobacteraceae to employ an oxygen-labile nitrogenase under aerobic conditions has been attributed to a high metabolic rate, allowing oxygen reduction at the cell membrane, the effectiveness of such a mechanism has been questioned at oxygen concentrations above 70 μM (ambient concentration is 230 μM O2), as well as during additional nutrient limitations. [38]

In addition to dinitrogen reduction, nitrogenases also reduce protons to dihydrogen, meaning nitrogenase is also a dehydrogenase. A list of other reactions carried out by nitrogenases is shown below: [39] [40]

Vanadium nitrogenases have also been shown to catalyze the conversion of CO into alkanes through a reaction comparable to Fischer-Tropsch synthesis.

There are two types of bacteria that synthesize nitrogenase and are required for nitrogen fixation. These are:

  • Free-living bacteria (non-symbiotic), examples include:
      (blue-green algae)
  • Azotobacter
    • Rhizobium, associated with leguminous plants
    • Spirillum, associated with cereal grasses
    • Frankia

    The three subunits of nitrogenase exhibit significant sequence similarity to three subunits of the light-independent version of protochlorophyllide reductase that performs the conversion of protochlorophyllide to chlorophyll. This protein is present in gymnosperms, algae, and photosynthetic bacteria but has been lost by angiosperms during evolution. [44]

    Separately, two of the nitrogenase subunits (NifD and NifH) have homologues in methanogens that do not fix nitrogen e.g. Methanocaldococcus jannaschii. [45] Little is understood about the function of these "class IV" nif genes, [46] though they occur in many methanogens. In M. jannaschii they are known to interact with each other and are constitutively expressed. [45]

    As with many assays for enzyme activity, it is possible to estimate nitrogenase activity by measuring the rate of conversion of the substrate (N2) to the product (NH3). Since NH3 is involved in other reactions in the cell, it is often desirable to label the substrate with 15 N to provide accounting or "mass balance" of the added substrate. A more common assay, the acetylene reduction assay or ARA, estimates the activity of nitrogenase by taking advantage of the ability of the enzyme to reduce acetylene gas to ethylene gas. These gases are easily quantified using gas chromatography. [47] Though first used in a laboratory setting to measure nitrogenase activity in extracts of Clostridium pasteurianum cells, ARA has been applied to a wide range of test systems, including field studies where other techniques are difficult to deploy. For example, ARA was used successfully to demonstrate that bacteria associated with rice roots undergo seasonal and diurnal rhythms in nitrogenase activity, which were apparently controlled by the plant. [48]

    Unfortunately, the conversion of data from nitrogenase assays to actual moles of N2 reduced (particularly in the case of ARA), is not always straightforward and may either underestimate or overestimate the true rate for a variety of reasons. For example, H2 competes with N2 but not acetylene for nitrogenase (leading to overestimates of nitrogenase by ARA). Bottle or chamber-based assays may produce negative impacts on microbial systems as a result of containment or disruption of the microenvironment through handling, leading to underestimation of nitrogenase. Despite these weaknesses, such assays are very useful in assessing relative rates or temporal patterns in nitrogenase activity.


    Abstract

    The iron (Fe) proteins of molybdenum (Mo)-, vanadium (V)-, and iron (Fe)-only nitrogenases are encoded by nifH, vnfH, and anfH, respectively. While the nifH-encoded Fe protein has been extensively studied over recent years, information regarding the properties of the vnfH- and anfH-encoded Fe proteins has remained scarce. Here, we present a combined biochemical, electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) analysis of the [Fe4S4] clusters of NifH, VnfH, and AnfH of Azotobacter vinelandii. Our data show that all three Fe proteins contain [Fe4S4] clusters of very similar spectroscopic and geometric structural properties, although NifH differs more from VnfH and AnfH with regard to the electronic structure. These observations have an interesting impact on the theory of the plausible sequence of evolution of nitrogenase Fe proteins. More importantly, the results presented herein provide a platform for future investigations of the differential activities of the three Fe proteins in nitrogenase biosynthesis and catalysis.


    Abstract

    Molybdenum nitrogenase catalyzes the reduction of dinitrogen into ammonia, which requires the coordinated transfer of eight electrons to the active site cofactor (FeMoco) through the intermediacy of an [8Fe-7S] cluster (P-cluster), both housed in the molybdenum–iron protein (MoFeP). Previous studies on MoFeP from two different organisms, Azotobacter vinelandii (Av) and Gluconacetobacter diazotrophicus (Gd), have established that the P-cluster is conformationally flexible and can undergo substantial structural changes upon two-electron oxidation to the P OX state, whereby a backbone amidate and an oxygenic residue (Ser or Tyr) ligate to two of the cluster’s Fe centers. This redox-dependent change in coordination has been implicated in the conformationally gated electron transfer in nitrogenase. Here, we have investigated the role of the oxygenic ligand in Av MoFeP, which natively contains a Ser ligand (βSer188) to the P-cluster. Three variants were generated in which (1) the oxygenic ligand was eliminated (βSer188Ala), (2) the P-cluster environment was converted to the one in Gd MoFeP (βPhe99Tyr/βSer188Ala), and (3) two oxygenic ligands were simultaneously included (βPhe99Tyr). Our studies have revealed that the P-cluster can become compositionally labile upon oxidation and reversibly lose one or two Fe centers in the absence of the oxygenic ligand, while still retaining wild-type-like dinitrogen reduction activity. Our findings also suggest that Av and Gd MoFePs evolved with specific preferences for Ser and Tyr ligands, respectively, and that the structural control of these ligands must extend beyond the primary and secondary coordination spheres of the P-cluster. The P-cluster adds to the increasing number of examples of inherently labile Fe–S clusters whose compositional instability may be an obligatory feature to enable redox-linked conformational changes to facilitate multielectron redox reactions.


    Experimental Procedures

    Unless noted otherwise, all chemicals and reagents were obtained from Fisher Scientific or Sigma-Aldrich.

    Cell Growth and Protein Purification

    All Azobacter vinelandii strains were grown in 180 L batches in a 200 L New Brunswick fermentor in Burke’s minimal medium supplemented with 2 mM ammonium acetate. The growth rate was measured by cell density at 436 nm. After ammonia had been consumed, the cells were de-repressed for 3 h and subsequently harvested by using a flow-through centrifugal harvester (Cepa, Lahr/Schwarzwald, Germany). The cell paste was washed with 50 mM Tris-HCl (pH 8.0). The ΔnifH NifDK, ΔnifB NifDK, and wild-type NifH proteins used in this work were purified as described previously. 9,14

    Sample Preparation

    All MCD samples were prepared in an Ar-filled anaerobic chamber (Vacuum Atmospheres, Hawthorne, CA) at an oxygen level of τ ppm. 11 The Ti(III) citrate solution was prepared as described previously. 15 DTN-reduced protein samples were in 25 mM Tris-HCl (pH 8.0), 10% glycerol, and 2 mM dithionite (Na2S2O4). Ti(III) citrate-reduced protein samples were prepared by incubating protein with 12 mM Ti(III) citrate for 5 min and subsequently removing excess Ti(III) citrate with a G25 size-exclusion column. Indigo disulfonate (IDS)-oxidized protein samples were prepared by incubating samples with IDS for 5 min and subsequently removing excess IDS with a G25 size-exclusion column. Samples were then concentrated to � mg/mL in a Centricon-50 concentrator (Amicon) as described previously, 14 transferred to MCD sample cuvettes, and frozen in a liquid nitrogen/pentane slush. All samples contained 50% glycerol to ensure the formation of an optical glass upon freezing, and they were kept on dry ice during transit.

    MCD Spectroscopy

    MCD spectra were recorded with a modified CD spectropolarimeter (model J-715, Jasco) interfaced with a superconducting magnet (model 400-7T Spectromag, Oxford). Sample temperatures were monitored with two thin film resistance temperature sensors [model CX1050-Cu-1-4L (Lakeshore, Westerville, OH)] positioned directly (1 mm) above and below the sample cuvette. The linearity of the magnetic field was monitored with a calibrated Hall generator [model HGCA-3020 (Lakeshore)] placed directly outside the superconducting magnet.

    MCD sample cells were constructed from optical-quality Spectrosil quartz [170� nm, 1 mm path length, model BS-1-Q-1, Starna, model SUV R-1001 or FUV (Spectrocell, Oreland, PA)]. Each cuvette was cut into the appropriate dimensions to fit the sample holder (2.0 cm × 12.5 mm), resulting in a sample volume of approximately 160 μL.

    MCD spectra were recorded at a rate of 50 nm/min and a resolution of 10 nm. Two different photomultipliers were used, one with a spectral range of 200� nm and the other with a spectral range of 700� nm. Because of the strong absorbance of DTN, all of the spectra presented herein start at � nm. Because optical glasses formed at low temperatures often generate a strain-induced background CD spectrum, the CD spectrum was recorded in zero magnetic field to determine whether the background signal was excessive. To eliminate interference by any background CD signal, the corrected MCD spectrum was obtained for each sample by first recording the spectrum with the magnetic field in one direction and then subtracting from it the spectrum with the field in the opposite direction. All spectral intensities were corrected for path length and sample concentration.

    Analysis of Magnetization Data

    Magnetization curves were recorded at a set wavelength and temperature while the magnetic field was linearly varied from 0 to 6 T at a rate of 0.1 A/s with a resolution of 2 s. MCD magnetization data were analyzed by a previously published fit/simulation program. 16 The program allows the calculation of best-fit saturation magnetization curves using experimental data as a basis set and is valid for any spin state, half-integer or integer, at any specified temperature.

    Experimental data were analyzed by fitting the Spin Hamiltonian parameters (g for S = 1 /2 and D and E/D for S > 1 /2) and the effective transition moment products, Mxy eff , Mxz eff , and Mxy eff , with a scaling parameter Asatlim = γ/4πS, where γ is the magnetogyric ratio. The effective transition moment products represent the planes of polarization that reflect the anisotropy of the g factors. Because the initial slope of the magnetization curve is dependent on the g factors, the transition polarizations relate the transition dipole to the g factor axes of a powder or randomly oriented sample.


    Results and Discussion

    Recently, we have shown that NifEN contains a Mo-free FeMoco precursor (15, 17), which can reconstitute and activate the FeMoco-deficient ΔnifB MoFe protein in a so-called “FeMoco maturation assay” (15). Such an assay contains the following: (i) NifEN, the source of FeMoco precursor (ii) Mo and homocitrate, the missing components from the precursor (iii) the Fe protein and MgATP, factors facilitating FeMoco maturation in an unknown fashion and (iv) ΔnifB MoFe protein, the receptor for FeMoco. Based on this assay, we developed a strategy to “uncouple” the original FeMoco maturation assay into several individual steps, allowing further determination of the sequence of events during FeMoco maturation and the roles of particular components in this process. Such a strategy includes the following steps: first, NifEN is incubated with molybdate, homocitrate, Fe protein, and MgATP then, His-tagged NifEN is repurified from the mixture by affinity chromatography and the nontagged wild-type Fe protein is repurified from the flow-through of the affinity column and finally, repurified components are tested for their capacities to reconstitute and activate the FeMoco-deficient ΔnifB MoFe protein. Analysis of repurified NifEN (designated NifEN complete ), therefore, enables the determination of the extent of maturation of the NifEN-bound precursor. Based on the study of NifEN complete , we have established that Mo and homocitrate are incorporated into the precursor while it is bound to NifEN (20). Meanwhile, analysis of the repurified Fe protein from the same incubation mixture (designated Fe protein complete ) allows the determination of whether Fe protein is indeed involved in Mo mobilization during FeMoco biosynthesis and, if so, what type of species it carries. Fe protein complete was examined by metal and spectroscopic analyses and tested for its capacity as (i) a “Mo/homocitrate donor” in an assay that consists of Fe protein complete , NifEN, and ΔnifB MoFe protein and (ii) a direct “FeMoco donor” in an assay that consists of only Fe protein complete and ΔnifB MoFe protein. Control experiments were conducted with Fe proteins repurified from incubation mixtures that lacked one or more of the maturation factors or contained (i) precursor-free ΔnifB NifEN instead of NifEN, (ii) ATP hydrolysis-deficient Fe protein variants instead of wild-type Fe protein, or (iii) ADP, or nonhydrolyzable ATP analogs, instead of ATP. Together with Fe protein complete , the repurified Fe proteins are categorically designated Fe proteins', with different superscripts indicating different conditions for sample preparation. For designations of Fe proteins', please refer to Materials and Methods.

    A Fe protein complete monomer, like that of the wild-type Fe protein, is ≈30 kDa (data not shown). The molecular mass of Fe protein complete is ≈60 kDa based on its elution profile on gel filtration Sephacryl S-200 high-resolution column (data not shown), indicating that Fe protein complete is a homodimer. Fe protein complete cannot serve as a direct FeMoco donor to the FeMoco-deficient ΔnifB MoFe protein because incubation of Fe protein complete with ΔnifB MoFe protein alone does not result in the reconstitution and activation of ΔnifB MoFe protein (Table 1). On the other hand, when Fe protein complete is incubated with NifEN and ΔnifB MoFe protein, ΔnifB MoFe protein is activated to approximately the same extent as it is by a complete FeMoco maturation assay or by incubation with NifEN complete (Table 1) (15, 20). Further, upon incubation with NifEN and increasing amounts of Fe protein complete , a maximum activity of ≈300 nmol of C2H4 formation per mg of ΔnifB MoFe protein per min is observed (Fig. 1 A). These observations indicate that Fe protein complete is competent in the maturation of the NifEN-bound FeMoco precursor without additional factors. Moreover, given that the precursor does not contain Mo and homocitrate and that no additional Mo and homocitrate are included in the assay, Fe protein complete appears to be the only source that can provide these two missing components for the transformation of the precursor into a fully assembled FeMoco. This argument is further supported by the observation that Fe protein minus Mo/homocitrate , Fe protein minus Mo , and Fe protein minus homocitrate are not competent in stimulating FeMoco maturation on NifEN (Table 1). These results strongly suggest that Fe protein acts as a Mo/homocitrate insertase that binds and delivers Mo and homocitrate to the Mo-free FeMoco precursor on NifEN.


    Combining a Nitrogenase Scaffold and a Synthetic Compound into an Artificial Enzyme

    Nitrogenase catalyzes substrate reduction at its cofactor center ([(Cit)MoFe7S9C](n-) designated M-cluster). Here, we report the formation of an artificial, nitrogenase-mimicking enzyme upon insertion of a synthetic model complex ([Fe6S9(SEt)2](4-) designated Fe6(RHH)) into the catalytic component of nitrogenase (designated NifDK(apo)). Two Fe6(RHH) clusters were inserted into NifDK(apo), rendering the conformation of the resultant protein (designated NifDK(Fe)) similar to the one upon insertion of native M-clusters. NifDK(Fe) can work together with the reductase component of nitrogenase to reduce C2H2 in an ATP-dependent reaction. It can also act as an enzyme on its own in the presence of Eu(II)DTPA, displaying a strong activity in C2H2 reduction while demonstrating an ability to reduce CN(-) to C1-C3 hydrocarbons in an ATP-independent manner. The successful outcome of this work provides the proof of concept and underlying principles for continued search of novel enzymatic activities based on this approach.

    Keywords: CC coupling artificial enzyme hydrocarbon nitrogenase synthetic compound.

    © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

    Figures

    Nitrogenase cofactor, synthetic compound and…

    Nitrogenase cofactor, synthetic compound and protein scaffold. Structural models of the M-cluster (…

    C 2 H 2 reduction by NifDK Fe in ATP-dependent and independent reactions.…


    Present address: Department of Chemistry, Tulane University, New Orleans, LA, USA

    Affiliations

    Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Alex McSkimming & Daniel L. M. Suess

    You can also search for this author in PubMed Google Scholar

    You can also search for this author in PubMed Google Scholar

    Contributions

    A.M. performed the experiments. A.M. and D.L.M.S. designed the research, analysed the data, and wrote the manuscript.

    Corresponding author