Exploring the Role of the Central Carbide of the Nitrogenase Active-Site FeMo-cofactor through Targeted 13C Labeling and ENDOR Spectroscopy.

Mo-dependent nitrogenase is a major contributor to global biological N2 reduction, which sustains life on Earth. Its multi-metallic active-site FeMo-cofactor (Fe7MoS9C-homocitrate) contains a carbide (C4-) centered within a trigonal prismatic CFe6 core resembling the structural motif of the iron carbide, cementite. The role of the carbide in FeMo-cofactor binding and activation of substrates and inhibitors is unknown. To explore this role, the carbide has been in effect selectively enriched with 13C, which enables its detailed examination by ENDOR/ESEEM spectroscopies. 13C-carbide ENDOR of the S = 3/2 resting state (E0) is remarkable, with an extremely small isotropic hyperfine coupling constant, Ca = +0.86 MHz. Turnover under high CO partial pressure generates the S = 1/2 hi-CO state, with two CO molecules bound to FeMo-cofactor. This conversion surprisingly leaves the small magnitude of the 13C carbide isotropic hyperfine-coupling constant essentially unchanged, Ca = -1.30 MHz. This indicates that both the E0 and hi-CO states exhibit an exchange-coupling scheme with nearly cancelling contributions to Ca from three spin-up and three spin-down carbide-bound Fe ions. In contrast, the anisotropic hyperfine coupling constant undergoes a symmetry change upon conversion of E0 to hi-CO that may be associated with bonding and coordination changes at Fe ions. In combination with the negligible difference between CFe6 core structures of E0 and hi-CO, these results suggest that in CO-inhibited hi-CO the dominant role of the FeMo-cofactor carbide is to maintain the core structure, rather than to facilitate inhibitor binding through changes in Fe-carbide covalency or stretching/breaking of carbide-Fe bonds.

Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants.

Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.

Purification of Nitrogenase Proteins.

A major hurdle in the studies of nitrogenase, one of the most complicated metalloenzymes known to date, is to obtain large amounts of intact, active proteins. Nitrogenase and related proteins are often multimeric and consist of metal centers that are critical for their activities. Most notably, the well-studied MoFe protein of Mo-nitrogenase is a heterotetramer that houses two of the most complicated metal clusters found in nature, the P-cluster and the FeMoco (or M-cluster). The structural complexity of these proteins and the oxygen sensitivity of their associated metal clusters, along with the demand for large amounts of high-quality proteins in most downstream analyses, make large-scale, high-yield purification of fully competent nitrogenase proteins a formidable task and yet, at the same time, a prerequisite for the success of nitrogenase research. This chapter highlights several methods that have been developed over the past few decades chiefly for the purification of naturally expressed nitrogenase in the diazotroph Azotobacter vinelandii. In addition, purification and Fe-S reconstitution strategies are also outlined for the heterologously expressed nitrogenase proteins in Escherichia coli.

Application of affinity purification methods for analysis of the nitrogenase system from Azotobacter vinelandii.

Nitrogenases are complex two-component metalloenzymes that catalyze biological nitrogen fixation. Three different nitrogenase types are found in the model nitrogen-fixing microbe Azotobacter vinelandii. In the case of the Mo-dependent enzyme, the two catalytic partners are referred to as the Fe protein and MoFe protein. In addition to genes encoding the catalytic components, there are a total of 68 other gene products known to be variously involved in producing, activating, protecting, sustaining, and regulating formation of the Mo-dependent nitrogenase. In order to support experiments designed to gain insight into the catalytic mechanism and assembly of nitrogenase, four different affinity-based purification protocols have been developed. These include an improved Co2+-based Immobilized Metal Affinity Chromatography (IMAC) method for the purification of MoFe protein, a newly developed StrepTactin Affinity Chromatography (STAC) method for the purification of MoFe protein and its assembly intermediates, a combined IMAC and STAC method for isolation of highly pure MoFe protein, and a STAC-based bait-prey method for isolation of complexes variously involved in the maturation process.

Kinetic Understanding of N2 Reduction versus H2 Evolution at the E4(4H) Janus State in the Three Nitrogenases.

The enzyme nitrogenase catalyzes the reduction of N2 to ammonia but also that of protons to H2. These reactions compete at the mechanistically central 'Janus' intermediate, denoted E4(4H), which has accumulated 4e-/4H+ as two bridging Fe-H-Fe hydrides on the active-site cofactor. This state can lose e-/H+ by hydride protonolysis (HP) or become activated by reductive elimination ( re) of the two hydrides and bind N2 with H2 loss, yielding an E4(2N2H) state that goes on to generate two NH3 molecules. Thus, E4(4H) represents the key branch point for these competing reactions. Here, we present a steady-state kinetic analysis that precisely describes this competition. The analysis demonstrates that steady-state, high-electron flux turnover overwhelmingly populates the E4 states at the expense of less reduced states, quenching HP at those states. The ratio of rate constants for E4(4H) hydride protonolysis ( kHP) versus reductive elimination ( kre) provides a sensitive measure of competition between these two processes and thus is a central parameter of nitrogenase catalysis. Analysis of measurements with the three nitrogenase variants (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase) reveals that at a fixed N2 pressure their tendency to productively react with N2 to produce two NH3 molecules and an accompanying H2, rather than diverting electrons to the side reaction, HP production of H2, decreases with their ratio of rate constants, k re/ kHP: Mo-nitrogenase, 5.1 atm-1; V-nitrogenase, 2 atm-1; and Fe-nitrogenase, 0.77 atm-1 (namely, in a 1:0.39:0.15 ratio). Moreover, the lower catalytic effectiveness of the alternative nitrogenases, with more H2 production side reaction, is not caused by a higher kHP but by a significantly lower k re.

Radical S-Adenosyl-l-Methionine (SAM) Enzyme Involved in the Maturation of the Nitrogenase Cluster.

Nitrogenase is the only known enzymatic system that converts atmospheric dinitrogen (N2) into bioavailable ammonia (NH3). The active-site cofactor responsible for this reactivity is a [(R-homocitrate)MoFe7S9C] cluster that is designated as the M-cluster. This important cofactor is assembled stepwise from a pair of [Fe4S4] clusters that become fused into a [Fe8S9C] core before additional refinements take place to complete the biosynthesis. NifB, a member of the radical S-adenosyl-l-methionine (SAM) superfamily, facilitates the conversion of the [Fe4S4] clusters (called the K-cluster) to the [Fe8S9C] core (called the L-cluster). This transformation includes a SAM-dependent carbide insertion with concomitant incorporation of an additional sulfur. While difficulties with the purification of NifB have historically prevented detailed biochemical analyses, we have developed a heterologous expression system in Escherichia coli that yields stable NifB proteins from various N2-fixing methanogenic organisms that can be used for studies. This chapter details the procedures necessary to prepare an active NifB protein. The methods used for the biochemical characterization of the SAM-dependent carbide insertion reactions are also described.

Crystal structure of VnfH, the iron protein component of vanadium nitrogenase.

Nitrogenases catalyze the biological fixation of inert N2 into bioavailable ammonium. They are bipartite systems consisting of the catalytic dinitrogenase and a complementary reductase, the Fe protein that is also the site where ATP is hydrolyzed to drive the reaction forward. Three different subclasses of dinitrogenases are known, employing either molybdenum, vanadium or only iron at their active site cofactor. Although in all these classes the mode and mechanism of interaction with Fe protein is conserved, each one encodes its own orthologue of the reductase in the corresponding gene cluster. Here we present the 2.2 Å resolution structure of VnfH from Azotobacter vinelandii, the Fe protein of the alternative, vanadium-dependent nitrogenase system, in its ADP-bound state. VnfH adopts the same conformation that was observed for NifH, the Fe protein of molybdenum nitrogenase, in complex with ADP, representing a state of the functional cycle that is ready for reduction and subsequent nucleotide exchange. The overall similarity of NifH and VnfH confirms the experimentally determined cross-reactivity of both ATP-hydrolyzing reductases.

Conformationally Gated Electron Transfer in Nitrogenase. Isolation, Purification, and Characterization of Nitrogenase From Gluconacetobacter diazotrophicus.

Nitrogenase is a complex, bacterial enzyme that catalyzes the ATP-dependent reduction of dinitrogen (N2) to ammonia (NH3). In its most prevalent form, it consists of two proteins, the catalytic molybdenum-iron protein (MoFeP) and its specific reductase, the iron protein (FeP). A defining feature of nitrogenase is that electron and proton transfer processes linked to substrate reduction are synchronized by conformational changes driven by ATP-dependent FeP-MoFeP interactions. Yet, despite extensive crystallographic, spectroscopic, and biochemical information on nitrogenase, the structural basis of the ATP-dependent synchronization mechanism is not understood in detail. In this chapter, we summarize some of our efforts toward obtaining such an understanding. Experimental investigations of the structure-function relationships in nitrogenase are challenged by the fact that it cannot be readily expressed heterologously in nondiazotrophic bacteria, and the purification protocols for nitrogenase are only known for a small number of diazotrophic organisms. Here, we present methods for purifying and characterizing nitrogenase from a new model organism, Gluconacetobacter diazotrophicus. We also describe procedures for observing redox-dependent conformational changes in G. diazotrophicus nitrogenase by X-ray crystallography and electron paramagnetic resonance spectroscopy, which have provided new insights into the redox-dependent conformational gating processes in nitrogenase.

Nitrogenase Assembly: Strategies and Procedures.

Nitrogenase is a metalloenzyme system that plays a critical role in biological nitrogen fixation, and the study of how its metallocenters are assembled into functional entities to facilitate the catalytic reduction of dinitrogen to ammonia is an active area of interest. The diazotroph Azotobacter vinelandii is especially amenable to culturing and genetic manipulation, and this organism has provided the basis for many insights into the assembly of nitrogenase proteins and their respective metallocofactors. This chapter will cover the basic procedures necessary for growing A. vinelandii cultures and subsequent recombinant transformation and protein expression techniques. Furthermore, protocols for nitrogenase protein purification and substrate reduction activity assays are described. These methods provide a solid framework for the assessment of nitrogenase assembly and catalysis.

Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion.

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.

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

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.

Purification of nitrogenase proteins.

Nitrogenase is one of the most complex enzymes known to date. The extensively studied molybdenum nitrogenase consists of two protein components and three metal centers that are critical for nitrogenase activity. The inherent complexity of this enzyme system, which is further compounded by the sensitivity of the metal clusters toward oxygen, makes the large-scale purification of fully active nitrogenase proteins a formidable task. This chapter highlights several methods that have been developed for the purification of nitrogenase proteins over the past few decades. Techniques used include weak anion exchange chromatography, size exclusion chromatography, and immobilized metal affinity chromatography. These methods can be selectively applied to nitrogenase variants and other related proteins.

Characterization of isolated nitrogenase FeVco.

The cofactors of the Mo- and V-nitrogenases (i.e., FeMoco and FeVco) are homologous metal centers with distinct catalytic properties. So far, there has been only one report on the isolation of FeVco from Azotobacter chroococcum. However, this isolated FeVco species did not carry the full substrate-reducing capacity, as it is unable to restore the N(2)-reducing ability of the cofactor-deficient MoFe protein. Here, we report the isolation and characterization of a fully active species of FeVco from A. vinelandii. Our metal and activity analyses show that FeVco has been extracted intact, carrying with it the characteristic capacity to reduce C(2)H(2) to C(2)H(6) and, perhaps even more importantly, the ability to reduce N(2) to NH(3). Moreover, our EPR and XAS/EXAFS investigations indicate that FeVco is similar to, yet distinct from FeMoco in electronic properties and structural topology, which could account for the differences in the reactivity of the two cofactors. The outcome of this study not only permits the proposal of the first EXAFS-based structural model of the isolated FeVco but also lays a foundation for future catalytic and structural investigations of this unique metallocluster.

Unique features of the nitrogenase VFe protein from Azotobacter vinelandii.

Nitrogenase is an essential metalloenzyme that catalyzes the biological conversion of dinitrogen (N(2)) to ammonia (NH(3)). The vanadium (V)-nitrogenase is very similar to the "conventional" molybdenum (Mo)-nitrogenase, yet it holds unique properties of its own that may provide useful insights into the general mechanism of nitrogenase catalysis. So far, characterization of the vanadium iron (VFe) protein of Azotobacter vinelandii V-nitrogenase has been focused on 2 incomplete forms of this protein: alphabeta(2) and alpha(2)beta(2), both of which contain the small delta-subunit in minor amounts. Although these studies provided important information about the V-dependent nitrogenase system, they were hampered by the heterogeneity of the protein samples. Here, we report the isolation and characterization of a homogeneous, His-tagged form of VFe protein from A. vinelandii. This VFe protein has a previously-unsuspected, alpha(2)beta(2)delta(4)-heterooctameric composition. Further, it contains a P-cluster that is electronically and, perhaps, structurally different from the P-cluster of molybdenum iron (MoFe) protein. More importantly, it is catalytically distinct from the MoFe protein, particularly with regard to the mechanism of H(2) evolution. A detailed EPR investigation of the origins and interplays of FeV cofactor- and P-cluster-associated signals is presented herein, which lays the foundation for future kinetic and structural analysis of the VFe protein.

Molecular insights into nitrogenase FeMo cofactor insertion: the role of His 362 of the MoFe protein alpha 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.

FeMo cofactor maturation on NifEN.

FeMo cofactor (FeMoco) biosynthesis is one of the most complicated processes in metalloprotein biochemistry. Here we show that Mo and homocitrate are incorporated into the Fe/S core of the FeMoco precursor while it is bound to NifEN and that the resulting fully complemented, FeMoco-like cluster is transformed into a mature FeMoco upon transfer from NifEN to MoFe protein through direct protein-protein interaction. Our findings not only clarify the process of FeMoco maturation, but also provide useful insights into the other facets of nitrogenase chemistry.

Vanadium (V) is reduced by the 'as isolated' nitrogenase Fe-protein at neutral pH.

Orthovanadate has been investigated in the presence of the nitrogenase Fe-protein. Electron paramagnetic resonance (EPR) spectra demonstrate that vanadium (V) is reduced by the reduced Fe-protein to vanadium (IV) which then probably binds to the nucleotide binding site in place of the Mg2+ which is normally present. In contrast, the oxidized Fe-protein is unable to reduce vanadate. In this case vanadate has potential for use as a phosphate analogue where it acts as transition state mimic for hydrolysis.

Identification of nif genes in N2-fixing bacterial strains isolated from rice fields along the Yangtze River Plain.

The aim of this research was to identify nifH and nifHDKYE ' genes in twenty strains of N2-fixing heterotrophic bacteria isolated from rice fields in the Yangtze River Plain. Southern hybridization of the total DNA from each strain was performed with the Klebsiella pneumoniae nifHDKYE ' gene probe (6.2 kb Eco RI fragment from pSA30) and the Azospirillum brasilense nifH gene probe (0.6 kb Eco RI-Hin dIII fragment from pHU8). We found that Eco RI fragments of total DNA from Aeromonas hydrophila HY2, Bacillus azotoformans FD, Bacillus licheniformis NCH1, NCH5, WH4, Bacillus brevis NC2, Bacillus pumilus NC12, Bacillus cereus NCH2, Citrobacter freundii HY5, HY9, Derxia gummosa HZ5, Pseudomonas mendocina HZ1 and Pseudomonas pseudoalcaligenes WH3 were positively hybridized with both of the probes. Agrobacterium radiobacter HY17, Corynebacterium sp. HY12, YZ and Pseudomonas sp. HY11 had Eco RI fragments hybridized with the K. pneumoniae nifHDKYE ' gene probe. An Eco RI fragment of total DNA from Bacillus megaterium YY4 was positively hybridized to the A. brasilense nifH gene probe. No hybridization sign was found in the total DNA fragments from Alcaligenes cupidus YY6 and Corynebacterium sp. NC11 hybridized with either of the gene probes. The data provide the number and size of EcoRI fragments of the total DNA hybridized with the nif gene probes for these strains of rarely studied species, suggesting additional evidence for N2 fixing and nif gene diversity of N2-fixing bacteria in rice fields along the Yangtze River Plain.

Nitrogenase proteins from Gluconacetobacter diazotrophicus, a sugarcane-colonizing bacterium.

Gluconacetobacter diazotrophicus Pal-5 grew well and expressed nitrogenase activity in the absence of NH4+ and at initial O2 concentrations greater than 5% in the culture atmosphere. G. diazotrophicus nitrogenase consisted of two components, Gd1 and Gd2, which were difficult to separate but were purified individually to homogeneity. Their compositions were very similar to those of Azotobacter vinelandii nitrogenase, however, all subunits were slightly smaller in size. The purified Gd1 protein contained a 12:1 Fe/Mo ratio as compared to 14:1 found for Av1 purified in parallel. Both Gd2 and Av2 contained 3.9 Fe atoms per molecule. Dithionite-reduced Gd1 exhibited EPR features at g=3.69, 3.96, and 4.16 compared with 3.64 and 4.27 for Av1. Gd2 gave an S=1/2 EPR signal identical to that of Av2. A Gd1 maximum specific activity of 1600 nmol H2 (min mg of protein)(-1) was obtained when complemented with either Gd2 or Av2, however, more Av2 was required. Gd2 had specific activities of 600 and 1100 nmol H2 (min mg protein)(-1) when complemented with Av1 and Gd1, respectively. The purified G. diazotrophicus nitrogenase exhibited a narrowed pH range for effective catalysis compared to the A. vinelandii nitrogenase, however, both exhibited maximum specific activity at about pH 7. The Gd-nitrogenase was more sensitive to ionic strength than the Av-nitrogenase.

Purification and characterization of NafY (apodinitrogenase gamma subunit) from Azotobacter vinelandii.

The formation of an active dinitrogenase requires the synthesis and the insertion of the iron-molybdenum cofactor (FeMo-co) into a presynthesized apodinitrogenase. In Azotobacter vinelandii, NafY (also known as gamma protein) has been proposed to be a FeMo-co insertase because of its ability to bind FeMo-co and apodinitrogenase. Here we report the purification and biochemical characterization of NafY and reach the following conclusions. First, NafY is a 26-kDa monomeric protein that binds one molecule of FeMo-co with very high affinity (K(d) approximately equal to 60 nm); second, the NafY-FeMo-co complex exhibits a S = 3/2 EPR signal with features similar to the signals for extracted FeMo-co and the M center of dinitrogenase; third, site-directed mutagenesis of nafY indicates that the His(121) residue of NafY is involved in cofactor binding; and fourth, NafY binding to apodinitrogenase or to FeMo-co does not require the presence of any additional protein. In addition, we have obtained evidence that suggests the ability of NafY to bind NifB-co, an FeS cluster of unknown structure that is a biosynthetic precursor to FeMo-co.