Molybdenum-iron Protein Research Articles

A voltammetric study of nitrogenase MoFe-protein using low-potential electron transfer mediators

The molybdenum-iron protein (MoFeP), a component of the enzyme nitrogenase, catalyzes the reduction of an array of small molecules, including N2 to NH3. In microorganisms, during the catalytic cycle, MoFeP receives electrons from the obligate biological redox partner iron protein (FeP) in a process coupled to the hydrolysis of two MgATP per one electron transferred. Despite the favorable redox properties of the cofactors, the requirement of the MgATP hydrolysis significantly decreases the energy efficiency of MoFeP. Therefore, remarkable efforts have been devoted to electrochemically activating MoFeP without FeP and MgATP. Previously, MoFeP was adsorbed on an electrode surface and revealed a slow catalysis with and without electron transfer mediators. However, enzyme adsorption can cause conformational and structural changes in a fragile protein molecule and alter its catalytic activity. In this work, MoFeP was electrochemically studied in solution. Various electron transfer mediators with potentials ranging from −0.3 V to −1 V (vs. NHE) were examined with MoFeP using cyclic voltammetry. No significant catalytic activity of the MoFeP was observed with any of the tested mediators. This indicates that efficient electrochemical activation of MoFeP cannot be achieved exclusively by increasing the driving force between the MoFeP redox cofactors and an electron donor.

Increased Nitrogenase Activity in Solar-Driven Biohybrids Containing Non-photosynthetic Bacteria and Conducting Polymers.

A conductive polymer-based photosynthetic biohybrid is constructed to enhance biological nitrogen fixation by increasing nitrogenase activity in the non-photosynthetic bacterium Azotobacter Chroococcum (A. Chroococcum). The light-harvesting cationic poly(fluorene-alt-phenylene) (PFP) electrostatically binds to the surface of the bacteria and possesses satisfactory conductivity to facilitate electron transfer to the bacterium, promoting the nitrogen fixation pathway through redox proteins on the surface of the bacteria when under illumination. Therefore, the nitrogenase activity, hydrogen, NH4 + -N and L-amino acids production are increased by 260 %, 37 %, 44 %, and 47 %, respectively. The expression levels of nifD and nifK encoding molybdenum-iron (MoFe) protein and relevant nitrogen-fixing proteins are up-regulated. These photoactive conductive polymer-bacteria biohybrids provide a new method for improving the biological nitrogen fixation capability of non-photosynthetic nitrogen-fixing bacteria.

β-N-Methylamino-L-Alanine (BMAA) Causes Severe Stress in Nostoc sp. PCC 7120 Cells under Diazotrophic Conditions: A Proteomic Study.

Non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is synthesized by cyanobacteria, diatoms, and dinoflagellates, and is known to be a causative agent of human neurodegenerative diseases. Different phytoplankton organisms’ ability to synthesize BMAA could indicate the importance of this molecule in the interactions between microalgae in nature. We were interested in the following: what kinds of mechanisms underline BMAA’s action on cyanobacterial cells in different nitrogen supply conditions. Herein, we present a proteomic analysis of filamentous cyanobacteria Nostoc sp. PCC 7120 cells that underwent BMAA treatment in diazotrophic conditions. In diazotrophic growth conditions, to survive, cyanobacteria can use only biological nitrogen fixation to obtain nitrogen for life. Note that nitrogen fixation is an energy-consuming process. In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by using LC-MS/MS spectrometry. Among them, 123 proteins belonging to different functional categories were selected—due to their notable expression differences—for further functional analysis and discussion. The presented proteomic data evidences that BMAA treatment leads to very strong (up to 80%) downregulation of α (NifD) and β (NifK) subunits of molybdenum-iron protein, which is known to be a part of nitrogenase. This enzyme is responsible for catalyzing nitrogen fixation. The genes nifD and nifK are under transcriptional control of a global nitrogen regulator NtcA. In this study, we have found that BMAA impacts in a total of 22 proteins that are under the control of NtcA. Moreover, BMAA downregulates 18 proteins that belong to photosystems I or II and light-harvesting complexes; BMAA treatment under diazotrophic conditions also downregulates five subunits of ATP synthase and enzyme NAD(P)H-quinone oxidoreductase. Therefore, we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as starvation-inducible DNA-binding protein, four SOS-response enzymes, and DNA repair enzymes, nine stress-response enzymes, and four proteases. The presented data provide new leads into the ecological impact of BMAA on microalgal communities that can be used in future investigations.

Draconibacterium mangrovi sp. nov., isolated from mangrove sediment.

A marine bacterial strain, designated GM2-18T, was isolated from mangrove sediment sampled at Luoyang River estuary, Quanzhou, PR China. Cells were Gram-stain-negative, slightly curved long rod-shaped and facultatively anaerobic with no flagellum. Catalase activity was found to be weak-positive and oxidase-positive. It had no ability to degrade or hydrolyse substrates including skimmed milk, cellulose, starch and Tweens (40, 60 and 80). The 16S rRNA gene sequence of strain GM2-18T had maximum similarity values to 'Draconibacterium filum' F2T, Draconibacterium sediminis JN14CK-3T and Draconibacterium orientale FH5T of 98.0, 97.8 and 97.4 %, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain GM2-18T was affiliated to the genus Draconibacterium and formed a clade with an uncultured bacterium clone identified from mangrove environment. Average nucleotide identity values and DNA-DNA hybridization estimates of strain GM2-18T compared to its Draconibacterium relatives strongly supported that it belonged to a new species. The respiratory quinone was menaquinone MK-7. The major fatty acids (>10 %) consisted of iso-C15 : 0, anteiso-C15 : 0 and C17 : 1 ω6c. The polar lipids were phosphatidylethanolamine, a phospholipid and several unidentified lipids. The genomic size of strain GM2-18T was 5.9 Mb and the G+C content was 40.8 mol%. Gene prediction and annotation of strain GM2-18T indicated that there was a nitrogen-fixing gene cluster encoding nitrogenase molybdenum-iron protein and related proteins responsible for nitrogen fixation. Based on the above characteristics, strain GM2-18T represents a novel species within the genus Draconibacterium. Thus, Draconibacterium mangrovi sp. nov. is proposed with type strain GM2-18T (=MCCC 1K04382T=KCTC 72879T), isolated from mangrove sediment.

Redox-Dependent Metastability of the Nitrogenase P-Cluster.

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 POX 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.

Structural characterization of the P1+ intermediate state of the P-cluster of nitrogenase

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3 The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2+, P1+, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.

Structural characterization of the nitrogenase molybdenum-iron protein with the substrate acetylene trapped near the active site

The biological reduction of dinitrogen (N2) to ammonia is catalyzed by the complex metalloenzyme nitrogenase. Structures of the nitrogenase component proteins, Iron (Fe) protein and Molybdenum‑iron (MoFe) protein, and the stabilized complexes these component proteins, have been determined, providing a foundation for a number of fundamental aspects of the complicated catalytic mechanism. The reduction of dinitrogen to ammonia is a complex process that involves the binding of N2 followed by reduction with multiple electrons and protons. Electron transfer into nitrogenase is typically constrained to the unique electron donor, the Fe protein. These constraints have prevented structural characterization of the active site with bound substrate. Recently it has been realized that selected amino acid substitutions in the environment of the active site metal cluster (Iron‑molybdenum cofactor, FeMo-co) allow substrates to persist even in the resting state. Reported here is a 1.70Å crystal structure of a nitrogenase MoFe protein α-96Arg➔Gln variant with the alternative substrate acetylene trapped in a channel in close proximity to FeMo-co. Complementary theoretical calculations support the validity of the acetylene interaction at this site and is also consistent with more favorable interactions in the variant MoFe protein compared to the native MoFe protein. This work represents the first structural evidence of a substrate trapped in the nitrogenase MoFe protein and is consistent with earlier assignments of proposed substrate pathways and substrate binding sites deduced from biochemical, spectroscopic, and theoretical studies.

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.

How does nitrogenase spring its trap?

Biochemistry It's a bit of a chemical mystery why, after eons of evolution to optimize efficiency, nitrogenase enzymes still make hydrogen as a by-product when they make ammonia. Recent work suggests that this step is key to binding the nitrogen molecule in the active site. Khadka et al. attached the molybdenum-iron protein of nitrogenase to an electrode to isolate the kinetics of hydrogen production. By measuring deuterium isotope effects and performing accompanying density functional theory calculations, they established that the rate-limiting step involves formation of one hydrogen molecule by proton transfer from sulfur to a bridging iron hydride. J. Am. Chem. Soc. 10.1021/jacs.7b07311 (2017).

Microcosm investigation of growth and phytoremediation potential of Azolla japonica along nitrogen gradients

ABSTRACTAlthough Azolla species are among the most promising plants for use in phytoremediation, more studies on their growth and nitrogen (N) uptake along the N gradients of growing media are required. In this study, N concentration-dependent growth in growing media and phosphorus (P) and N accumulation by Azolla japonica were studied by estimating direct N uptake from media by molybdenum-iron proteins. The doubling time of A. japonica was less than a week, regardless of the N concentration (0, 5, and 25 mg N/L) present in the growth media, indicating that this plant is suitable for remediation. Plants showed a high uptake of P, probably via plant-bacteria symbiosis, indicating their potential for effective P remediation. A. japonica also showed more than 4% N content regardless of the treatment and accumulated more than 40 mg of N per microcosm in 3 weeks. iron and molybdenum levels in plants were strongly associated with N fixation, and N uptake from media was estimated to be more than 25 mg per microcosm in 3 weeks, indicating that A. japonica has N remediation potential. As A. japonica is a rapidly growing plant, capable of efficient P and N remediation, it has great potential for use in phytoremediation of nutrient-enriched waters such as agricultural or urban wastewater and eutrophicated aquatic ecosystems.

Reversible Protonated Resting State of the Nitrogenase Active Site.

Protonated states of the nitrogenase active site are mechanistically significant since substrate reduction is invariably accompanied by proton uptake. We report the low pH characterization by X-ray crystallography and EPR spectroscopy of the nitrogenase molybdenum iron (MoFe) proteins from two phylogenetically distinct nitrogenases (Azotobacter vinelandii, Av, and Clostridium pasteurianum, Cp) at pHs between 4.5 and 8. X-ray data at pHs of 4.5–6 reveal the repositioning of side chains along one side of the FeMo-cofactor, and the corresponding EPR data shows a new S = 3/2 spin system with spectral features similar to a state previously observed during catalytic turnover. The structural changes suggest that FeMo-cofactor belt sulfurs S3A or S5A are potential protonation sites. Notably, the observed structural and electronic low pH changes are correlated and reversible. The detailed structural rearrangements differ between the two MoFe proteins, which may reflect differences in potential protonation sites at the active site among nitrogenase species. These observations emphasize the benefits of investigating multiple nitrogenase species. Our experimental data suggest that reversible protonation of the resting state is likely occurring, and we term this state “E0H+”, following the Lowe–Thorneley naming scheme.

Mutagenesis at α-423Ile of MoFe protein reduces the catalytic activity of nitrogenase in Klebsiella oxytoca

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia, the central process of biological nitrogen fixation. The mechanism of electron transfer from the P-cluster to the iron-molybdenum cofactor (FeMo-co) during the process of substrate reduction remains unclear. Based on our previous hypothesis, the dual pathway model, three mutants of Klebsiella oxytoca strain M5al (termed Nβ102A, Kα431H and Iα423P) were constructed to map the H+/e− transfer pathway, in which β-102Asn, α-431Lys and α-423Ile of the molybdenum-iron (MoFe) protein of nitrogenases were replaced by alanine, histidine and proline, respectively. All three substitutions affected the diazotrophic growth, especially of the Iα423P strain with very low capability of diazotrophic growth and C2H2 reduction activity. In a 42-L fermenter, the Iα423P strain was induced to the maximal C2H2 reduction activity, which was only ~17 % of that of M5al. However, the transcription level of the nifD gene was approximately four-fold higher than that of the M5al strain, indicating that the low C2H2 reduction activity of Iα423P was not because of downregulation of nifD expression. The maximal C2H2 and H+ reduction activities of the Iα423P MoFe protein were 13 % and 21 % of those of the wild-type MoFe protein, respectively. In conclusion, we propose that the changed properties of the Iα423P MoFe protein are related to the disruption of hydrogen bond formation between α-423Ile and homocitrate, and that α-423Ile serves as a gate to the Mo site in FeMo-co during the H+/e− transfer in nitrogenase.

ATP-Uncoupled, Six-Electron Photoreduction of Hydrogen Cyanide to Methane by the Molybdenum–Iron Protein

A detailed study of the eight-electron/eight-proton catalytic reaction of nitrogenase has been hampered by the fact that electron and proton flow in this system is controlled by ATP-dependent protein-protein interactions. Recent studies have shown that it is possible to circumvent the dependence on ATP through the use of potent small-molecule reductants or light-driven electron injection, but success has been limited to two-electron reductions of hydrazine, acetylene, or protons. Here we show that a variant of the molybdenum-iron protein labeled with a Ru-photosensitizer can support the light-driven, six-electron catalytic reduction of hydrogen cyanide into methane and likely also ammonia. Our findings suggest that the efficiency of this light-driven system is limited by the initial one- or two-electron reduction of the catalytic cofactor (FeMoco) to enable substrate binding, but the subsequent electron-transfer steps into the FeMoco-bound substrate proceed efficiently.

Electron transfer and half-reactivity in nitrogenase

Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum-iron protein (MoFe protein) and a protein containing an [Fe4-S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.

Nitrogenase Assembly Pathway

![Figure][1] CREDIT: KAISER ET AL. A key step in the global nitrogen cycle is the reduction of atmospheric dinitrogen to ammonia by nitrogenase, a complex metalloenzyme. The catalytic component of this enzyme, the molybdenum-iron protein NifDK, comprises two unusual metalloclusters, the [

Steric Control of the Hi‐CO MoFe Nitrogenase Complex Revealed by Stopped‐Flow Infrared Spectroscopy

Here, we report the impact of substitution for the α-70Val residue of Azotobacter vinelandii nitrogenase molybdenum-iron (MoFe) protein on the so-called “hi-CO” CO bound complex as monitored in real-time by stopped-flow infra-red (SF-IR) spectroscopy. Nitrogenase is a bacterial metalloenzyme system whose physiological function is to catalyze the reduction of dinitrogen to ammonia [1, 2] with a concomitant reduction of 2H+ to H2 and hydrolysis of MgATP. X-ray crystallography on MoFe nitrogenase reveals the active-site FeMo-cofactor to be an unprecedented [Fe7S9MoX:homocitrate] cluster [3, 4], figure 1. However, simple inspection of this structure does not provide an obvious location for substrate binding or any indication of the subsequent mechanism for substrate reduction. Mechanisms focused on substrate binding to either Mo and Fe sites have been proposed as have combination approaches that involve migration of substrate-derived moieties between metal atoms during reduction [5–8].

Formation of a homocitrate-free iron-molybdenum cluster on NifEN: Implications for the role of homocitrate in nitrogenase assembly

Molybdenum (Mo)-dependent nitrogenase is a complex metalloprotein that catalyzes the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) at the molybdenum-iron cofactor (FeMoco) site of its molybdenum-iron (MoFe) protein component. Here we report the formation of a homocitrate-free, iron-molybdenum ("FeMo") cluster on the biosynthetic scaffold of FeMoco, NifEN. Such a NifEN-associated "FeMo" cluster exhibits EPR features similar to those of the NifEN-associated, fully-complemented "FeMoco", which originate from the presence of Mo in both cluster species; however, "FeMo" cluster and "FeMoco" display different temperature-dependent changes in the line shape and the signal intensity of their respective EPR features, which reflect the impact of homocitrate on the redox properties of these clusters. XAS/EXAFS analysis reveals that the Mo centers in both "FeMo" cluster and "FeMoco" are present in a similar coordination environment, although Mo in "FeMo" cluster is more loosely coordinated as compared to that in "FeMoco" with respect to the Mo-O distances in the cluster, likely due to the absence of homocitrate that normally serves as an additional ligand for the Mo in the cluster. Subsequent biochemical investigation of the "FeMo" cluster not only facilitates the determination of the sequence of events in the mobilization of Mo and homocitrate during FeMoco maturation, but also permits the examination of the role of homocitrate in the transfer of FeMoco between NifEN and MoFe protein. Combined outcome of these studies establishes a platform for future structural analysis of the interactions between NifEN and MoFe protein, which will provide useful insights into the mechanism of cluster transfer between the two proteins.

Dual functions of NifEN: insights into the evolution and mechanism of nitrogenase

Nitrogenase catalyzes the nucleotide-dependent conversion of dinitrogen to ammonia at the iron-molybdenum cofactor (FeMoco) center of its molybdenum-iron (MoFe) protein component. Biosynthesis of FeMoco is arguably one of the most complex processes in the field of bioinorganic chemistry, which involves the participation of a number of nif (nitrogen fixing) gene products. One key player in this process, NifEN (encoded by nifE and nifN), is homologous to the MoFe protein with regard to both the primary sequences and the types of the metal centers. Recently, an all-iron precursor has been identified on NifEN, which closely resembles the Fe/S core structure of the mature cofactor. Such a precursor-bound form of NifEN has not only served as an excellent platform for the investigation of FeMoco assembly, but also facilitated the examination of the capacity of NifEN as a catalytic homolog of MoFe protein. This perspective will focus on the recent advances toward elucidating the dual functions of NifEN in nitrogenase assembly and catalysis, and the insights afforded by these advances into the evolution and mechanism of nitrogenase.

Decoding the Nitrogenase Mechanism: The Homologue Approach

The (Mo)-nitrogenase is a complex metalloenzyme that catalyzes the key step in the global nitrogen cycle, the reduction of atmospheric dinitrogen (N(2)) to bioavailable ammonia (NH(3)), at the iron-molybdenum cofactor (FeMoco) site of its molybdenum-iron (MoFe) protein component. Despite the fundamental significance of biological nitrogen fixation and extensive studies over the past decades, the catalytic mechanism of nitrogenase has not been deciphered. One major challenge for the mechanistic study of nitrogenase is the redox versatility of its FeMoco center. The ability of FeMoco to shuttle between oxidation states in a rapid and unsynchronized manner results in a mixed oxidation state of the cofactor population during turnover. The substrate and the various intermediates can only interact with the FeMoco site in a transient manner, so it is extremely difficult to capture any substrate- or intermediate-bound form of nitrogenase for the direct examination of substrate-enzyme interactions during catalysis. In this Account, we describe the approach of identifying a partially "defective" nitrogenase homologue, one with a slower turnover rate, as a means of overcoming this problem. The NifEN protein complex serves as an ideal candidate for this purpose. It is an alpha(2)beta(2)-heterotetramer that contains cluster-binding sites homologous to those found in the MoFe protein: the "P-cluster site" at the interface of the alphabeta-subunit dimer, which accommodates a [Fe(4)S(4)]-type cluster; and the "FeMoco site" within the alpha-subunit, which houses an all-iron homologue to the FeMoco. Moreover, NifEN mimics the MoFe protein in catalysis: it is capable of reducing acetylene (C(2)H(2)) and azide (N(3)(-)) in an ATP- and iron (Fe) protein-dependent manner. However, NifEN is unable to reduce proton (H(+)) and N(2), and it is an inefficient enzyme with a restricted electron flux during the turnover. The extremely slow turnover rate of NifEN and the possible "synchronization" of its FeMoco homologue at a certain oxidation level permit the observation of a new S = 1/2 EPR signal upon turnover of C(2)H(2) by NifEN, which is analogous to the signal reported for a MoFe protein variant upon turnover of the same substrate. This result is exciting, because it suggests the possibility of naturally enriching a C(2)H(2)-bound form of NifEN for the successful crystallization of the first intermediate-bound nitrogenase homologue. On the other hand, the fact that NifEN represents a partially "defective" homologue of the MoFe protein makes it a promising mutational platform on which a functional MoFe protein equivalent may be reconstructed by introducing the missing features of MoFe protein step-by-step into NifEN. Such a strategy allows us to define the function of each feature and address questions such as the following: What is the function of P-cluster in catalysis? Are Mo and homocitrate the essential constituents of the cofactor in N(2) reduction? How does substrate accessibility affect the reactivity of the enzyme? This homologue approach could complement the mechanistic analysis of the nitrogenase MoFe protein, and information derived from both approaches will help achieve the ultimate goal of solving the riddle of biological nitrogen fixation.

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.