Electron-paramagnetic-resonance spectroscopy and related techniques in the study of nitrogenase.
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Adenosine Triphosphate↗
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An extrusion strategy for the FeMo cofactor from nitrogenase. Towards synthetic iron-sulfur proteins.
Iron-sulfur clusters are ubiquitous in biological systems, facilitating functions such as electron transfer (rubredoxins, ferredoxins, rieske centres), isomerization (aconitase) and small molecule activation such as dinitrogen reduction (nitrogenases). Of global importance and recently particular interest, is the iron-sulfur-containing iron-molybdenum cofactor (FeMoco) cluster that achieves the biological reduction of dinitrogen under mild conditions. This biologically unique cluster has proved difficult to investigate due to its extreme air sensitivity and the instability of the cluster's structural integrity, outside the protective protein matrix. Here, we report a model iron-sulfur cluster (Roussins black salt (NH(4))[Fe(4)S(3)(NO)(7)]) that has been used to achieve the first example of a metal cluster (guest) embedded within a pseudo-protein, cyclodextrin (host). The product formed is supramolecular, that is, it contained no covalent bonds and was stabilized by predominantly entropy effects. Formation of a 1 : 1 complex between the host and the guest was established for the iron-sulfur cluster with either seven- or eight-membered cyclodextrins (beta- or gamma-cyclodextrin). A range of techniques was used to characterize the new complexes in both the solid and solution states. Electrospray mass spectra indicated the presence of parent ions of the host-guest complexes and electrochemistry was also used to define the redox behavior of the complexes. The iron-sulfur clusters were significantly more stable in the presence of the host cyclodextrin, as revealed by a negative shift for the reduction potential for the host-guest product. Using the beta-cyclodextrin as host, the reduction potential of the iron-sulfur cluster shifted more negative by 60 mV; the effect was even more dramatic for the larger gamma-cyclodextrin where the reduction potential for the cluster was shifted by 90 mV more negative than the 'unbound' [Fe(4)S(3)(NO)(7)]- cluster. This is the first example of a metal cluster, stabilized as a supramolecular complex in a 'host' environment outside of a covalently bonded protein matrix. Creating such stable environments for metal cofactors or clusters that otherwise spontaneously degrade or are catalytically inactive outside the protein matrix could have enormous practical value. Specific implications for the development of extrusion methods for FeMoco from nitrogenase are enormous, with previously difficult, high-energy molecular transformations, such as dinitrogen to ammonia, now more realistically accessible.
Crystallography, X-Ray↗
FeMo cofactor biosynthesis in a nifE- mutant of Rhodobacter capsulatus.
In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM). Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.
Acetylene↗
Activation and protonation of dinitrogen at the FeMo cofactor of nitrogenase.
The protonation of N2 bound to the active center of nitrogenase has been investigated using state-of-the-art density-functional theory calculations. Dinitrogen in the bridging mode is activated by forming two bonds to Fe sites, which results in a reduction of the energy for the first hydrogen transfer by 123 kJ/mol. The axial binding mode with open sulfur bridge is less reactive by 30 kJ/mol and the energetic ordering of the axial and bridged binding modes is reversed in favor of the bridging dinitrogen during the first protonation. Protonation of the central ligand is thermodynamically favorable but kinetically hindered. If the central ligand is protonated, the proton is transferred to dinitrogen following the second protonation. Protonation of dinitrogen at the Mo site does not lead to low-energy intermediates.
Binding Sites↗
Identification of a nitrogenase FeMo cofactor precursor on NifEN complex.
The biosynthesis of the FeMo cofactor (FeMoco) of Azotobacter vinelandii nitrogenase presumably starts with the production of its Fe/S core by NifB (the nifB gene product). This core is subsequently processed on the alpha2beta2 tetrameric NifEN complex (formed by the nifE and nifN gene products). In this article, we identify a NifEN-bound FeMoco precursor form that can be converted to fully assembled FeMoco in a so-called FeMoco-maturation assay containing only purified components. We also establish that only molybdate, homocitrate, MgATP, and Fe protein are essential for FeMoco maturation. The FeMoco-maturation assay described here will further address the remaining questions related to the assembly mechanism of the ever-intriguing FeMoco.
Azotobacter vinelandii↗
Nitrogenase reactivity with P-cluster variants.
Nitrogenase is a multicomponent metalloenzyme that catalyzes the conversion of atmospheric dinitrogen to ammonia. For decades, it has been generally believed that the [8Fe-7S] P-cluster of nitrogenase component 1 is indispensable for nitrogenase activity. In this study, we identified two catalytically active P-cluster variants by activity assays, metal analysis, and EPR spectroscopic studies. Further, we showed that both P-cluster variants resemble [4Fe-4S]-like centers based on x-ray absorption spectroscopic experiments. We believe that our findings challenge the dogma that the standard P-cluster is the only cluster species capable of supporting substrate reduction at the FeMo cofactor and provide important insights into the general mechanism of nitrogenase catalysis and assembly.
Azotobacter vinelandii↗
NifB-dependent in vitro synthesis of the iron-molybdenum cofactor of nitrogenase.
Biological nitrogen fixation, an essential process of the biogeochemical nitrogen cycle that supports life on Earth, is catalyzed by the nitrogenase enzyme. The nitrogenase active site contains an iron and molybdenum cofactor (FeMo-co) composed of 7Fe-9S-Mo-homocitrate and one not-yet-identified atom, which probably is the most complex [Fe-S] cluster in nature. Here, we show the in vitro synthesis of FeMo-co from its simple constituents, Fe, S, Mo, and homocitrate. The in vitro FeMo-co synthesis requires purified NifB and depends on S-adenosylmethionine, indicating that radical chemistry is required during FeMo-co assembly.
Azotobacter vinelandii↗
A methyldiazene (HN=N-CH3)-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism.
Methyldiazene (HN=N-CH3) isotopomers labeled with 15N at the terminal or internal nitrogens or with 13C or 2H were used as substrates for the nitrogenase alpha-195Gln-substituted MoFe protein. Freeze quenching under turnover traps an S = (1/2) state that has been characterized by EPR and 1H-, 15N-, and 13C-electron nuclear double resonance spectroscopies. These studies disclosed the following: (i) a methyldiazene-derived species is bound to the active-site FeMo cofactor; (ii) this species binds through an [-NHx] fragment whose N derives from the methyldiazene terminal N; and (iii) the internal N from methyldiazene probably does not bind to FeMo cofactor. These results constrain possible mechanisms for reduction of methyldiazene. In the Chatt-Schrock mechanism for N2 reduction, H atoms sequentially add to the distal N before N-N bond cleavage (d-mechanism). In a d-mechanism for methyldiazene reduction, a bound [-NHx] fragment only occurs after reduction by three electrons, which leads to N-N bond cleavage and the release of the first NH3. Thus, the appearance of bound [-NHx] is compatible with the d-mechanism only if it represents a late stage in the reduction process. In contrast are mechanisms where H atoms add alternately to distal and proximal nitrogens before N-N cleavage (a-mechanism) and release of the first NH3 after reduction by five electrons. An [-NHx] fragment would be bound at every stage of methyldiazene reduction in an a-mechanism. Although current information does not rule out the d-mechanism, the a-mechanism is more attractive because proton delivery to substrate has been specifically compromised in alpha-195Gln-substituted MoFe protein.
Binding Sites↗
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.
Electron Spin Resonance Spectroscopy↗
Nitrogenase Fe protein: A molybdate/homocitrate insertase.
The Fe protein is indispensable for nitrogenase catalysis and biosynthesis. However, its function in iron-molybdenum cofactor (FeMoco) biosynthesis has not been clearly defined. Here we show that the Fe protein can act as a Mo/homocitrate insertase that mobilizes Mo/homocitrate for the maturation of FeMoco precursor on NifEN. Further, we establish that Mo/homocitrate mobilization by the Fe protein likely involves hydrolysis of MgATP and protein-protein interaction between the Fe protein and NifEN. Our findings not only clarify the role of the Fe protein in FeMoco assembly and assign another function to this multitask enzyme but also provide useful insights into a mechanism of metal trafficking required for the assembly of complex metalloproteins such as nitrogenase.
Azotobacter vinelandii↗
The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase.
It is known that an E146D site-directed variant of the Azotobacter vinelandii iron protein (Fe protein) is specifically defective in its ability to participate in iron-molybdenum cofactor (FeMoco) insertion. Molybdenum-iron protein (MoFe protein) from the strain expressing the E146D Fe protein is partially ( approximately 45%) FeMoco deficient. The "free" FeMoco that is not inserted accumulates in the cell. We were able to insert this "free" FeMoco into the partially pure FeMoco-deficient MoFe protein. This insertion reaction required crude extract of the DeltanifHDK A. vinelandii strain CA12, Fe protein and MgATP. We used this as an assay to purify a required "insertion" protein. The purified protein was identified as GroEL, based on the molecular mass of its subunit (58.8 kDa), crossreaction with commercially available antibodies raised against E. coli GroEL, and its NH(2)-terminal polypeptide sequence. The NH(2)-terminal polypeptide sequence showed identity of up to 84% to GroEL from various organisms. Purified GroEL of A. vinelandii alone or in combination with MgATP and Fe protein did not support the FeMoco insertion into pure FeMoco-deficient MoFe protein, suggesting that there are still other proteins and/or factors missing. By using GroEL-containing extracts from a DeltanifHDK strain of A. vinelandii CA12 along with FeMoco, Fe protein, and MgATP, we were able to supply all required proteins and/or factors and obtained a fully active reconstituted E146D nifH MoFe protein. The involvement of the molecular chaperone GroEL in the insertion of a metal cluster into an apoprotein may have broad implications for the maturation of other metalloenzymes.
Amino Acid Sequence↗
Molybdenum cofactors from molybdoenzymes and in vitro reconstitution of nitrogenase and nitrate reductase.
A molybdenum cofactor (Mo-co) from xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) can be isolated from the enzyme by a technique that has been used to isolate an iron-molybdenum cofactor (FeMo-co) from component I of nitrogenase. N-Methylformamide is used for the extraction of these molybdenum cofactors. Mo-co from xanthine oxidase activates nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.2) in an extract from Neurospora crassa mutant strain Nit-1; however, FeMo-co is unable to activate nitrate reductase in strain Nit-1. Mo-co from xanthine oxidase is unable to activate nitrogenase in an extract of Azotobacter vinelandii mutant strain UW45. Inactive component I in this extract can be activated by FeMo-co. These results indicate that nitrate reductase and xanthine oxidase share a common molybdenum cofactor, but this cofactor is different from the molybdenum cofactor in nitrogenase.A. vinelandii synthesizes both Mo-co and FeMo-co. Mo-co is produced when the cells fix N(2) and also when they are repressed for nitrogenase synthesis by growth in a medium containing excess ammonium. However, FeMo-co is not produced when cells are grown in an ammonium-containing medium. Partially purified preparations of component I from A. vinelandii and Klebsiella pneumoniae contain both FeMo-co and Mo-co. The presence of both FeMo-co and Mo-co activities in partially purified preparations of component I explains previous reports of activation of inactive nitrate reductase in strain Nit-1 by acid-treated component I of nitrogenase. The Mo-co can be separated from FeMo-co in these preparations by chromatography on Sephadex G-100 in N-methylformamide. Both FeMo-co and Mo-co are sensitive to oxygen.
Azotobacter↗
Isolation of a molybdenum--iron cluster from nitrogenase.
A molybdenum-iron cluster (Mo-Fe cluster) containing 6 Fe atoms per Mo was isolated by methyl ethyl ketone extraction of component I of nitrogenase from Azotobacter vinelandii. The cluster has no EPR signal in the g = 4 region but has an intense signal at g = 2.05 and 2.01. After the cluster was transferred from methyl ethyl ketone to N-methylformamide, the signal in the g = 2 region disappeared and a signal similar to that found with Fe-Mo cofactor appeared. The Mo-Fe cluster is the EPR-active center that undergoes reversible oxidation-reduction during catalytic turnover at the active site of the enzyme. In contrast to the Fe-Mo cofactor, which contains 8 Fe atoms per Mo, the Mo-Fe cluster failed to activate either inactive component I in extracts of A. vinelandii mutant strain UW45 or tungsten-containing component I from wild-type A. vinelandii. On the other hand, the Mo-Fe cluster showed as much acetylene-reduction activity with sodium borohydride as the reductant as did the Fe-Mo cofactor. Like nitrogenase-dependent and Fe-Mo cofactor-dependent acetylene reduction, the Mo-Fe cluster-dependent acetylene reduction is strongly inhibited by carbon monoxide.
Azotobacter↗
Crystallographic properties of the MoFe proteins of nitrogenase from Clostridium pasteurianum and Azotobacter vinelandii.
Preliminary x-ray diffraction data from single crystals of the MoFe proteins of nitrogenase from Clostridium pasteurianum and Azotobacter vinelandii have been obtained. Both protein crystals belong to the P2(1) space group. The MoFe protein crystals from C. pasteurianum and A. vinelandii diffract to angles corresponding to resolutions as great as 2.4 A and 3.0 A, respectively. The cell dimensions of the MoFe protein crystals from the two species have been determined from precession photographs.
Azotobacter↗
H2-uptake activity of the MoFe protein component of Azotobacter vinelandii nitrogenase.
The MoFe protein from Azotobacter vinelandii catalyzes the reduction of methylene blue and other oxidants by H2 under anaerobic conditions. H2 uptake followed manometrically or by 3H2 transfer from the gas to aqueous phase occurs concomitantly with methylene blue disappearance monitored optically or coulometrically. The stoichiometry was found to be 1:1 methylene blue/H2. MoFe protein oxidized by transfer of approximately 4 e- seems to be the redox state of the protein most active in the catalytic step, although both the S2O4(2-)-reduced and 6-e- oxidized state have been shown to react, but at a much lower rate. The presence of H2 in the atmosphere above the MoFe protein offers increased protection against O2 inactivation.
Azotobacter↗
In vitro synthesis of the iron-molybdenum cofactor of nitrogenase.
Molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires the protein products of at least the nifB, nifN, and nifE genes. Extracts of FeMo-co-negative mutants of Klebsiella pneumoniae and Azotobacter vinelandii with lesions in different genes can be complemented for FeMo-co synthesis. Both K. pneumoniae and A. vinelandii dinitrogenase (component I) deficient in FeMo-co can be activated by FeMo-co synthesized in vitro. Properties of the partially purified dinitrogenase activated by FeMo-co synthesized in vitro were comparable to those of dinitrogenase from the wild-type organism; e.g., ratios of acetylene- to nitrogen-reduction activities, as well as those of acetylene reduction activities to EPR spectrum peak height at g = 3.65, were very similar. A. vinelandii mutants UW45 and CA30 have mutations in a gene functionally equivalent to nifB of K. pneumoniae.
Acetylene↗
Purification and characterization of the nifN and nifE gene products from Azotobacter vinelandii mutant UW45.
The nifN and -E gene products are involved in the synthesis of the iron-molybdenum cofactor of dinitrogenase, the enzyme responsible for the reduction of dinitrogen to ammonia. By using the in vitro iron-molybdenum cofactor biosynthesis assay, we have followed the purification of these gene products 450-fold to greater than 95% purity. An overall recovery of 20% was obtained with the purified protein having a specific activity of 6900 units/mg of protein. The protein (hereafter referred to as NIFNE) was found to contain equimolar amounts of the nifN and -E gene products and have a native molecular mass of 200 +/- 10 kDa, which indicates an alpha 2 beta 2 structure. NIFNE was oxygen labile with a half-life of 1 min in air. A UV-visible spectrum of the dye-oxidized protein showed an absorption maximum at 425 nm that could be bleached by reduction of NIFNE with sodium dithionite, suggesting the presence of an Fe center in NIFNE.
Azotobacter↗
Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor.
In vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase using homocitrate and its analogs allows the formation of modified forms of FeMo-co that show altered substrate specificities (N2, acetylene, cyanide, or proton reduction) of nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. The (1R,2S)-threo- and (1S,2S)-erythro-fluorinated diastereomers of homocitrate have been incorporated in vitro into dinitrogenase in place of homocitrate. Dinitrogenase activated with FeMo-co synthesized using threo-fluorohomocitrate reduces protons, cyanide, and acetylene but cannot reduce N2. In addition, proton reduction is inhibited by carbon monoxide (CO), a characteristic of dinitrogenase from NifV- mutants. Dinitrogenase activated with FeMo-co synthesized using erythro-fluorohomocitrate reduces protons, cyanide, acetylene, and N2. In this case proton reduction is not inhibited by CO, a characteristic of the wild-type enzyme. Cyanide reduction properties of dinitrogenase activated with FeMo-co containing either fluorohomocitrate diastereomer are similar, and CO strongly inhibits cyanide reduction. Dinitrogenases activated with FeMo-co containing homocitrate analogs with a hydroxyl group on the C-1 position are much less susceptible to CO inhibition of cyanide reduction. However, proton and cyanide reduction by dinitrogenase containing FeMo-co activated with (1R,2S) threo-isocitrate is only one-third that of dinitrogenase activated with the racemic mixture of -isocitrate and shows strong CO inhibition of substrate reduction. These results suggest that CO inhibition of proton and cyanide reduction occurs when the hydroxyl group on the C-1 position of analogs is "trans" to the C-2 carboxyl group (i.e., in the threo conformation). When racemic mixtures of these analogs are used in the system, it seems that the erythro form is preferentially incorporated into dinitrogenase. Finally, carbonyl sulfide inhibition of substrate reduction by dinitrogenase is dependent on the homocitrate analog incorporated into FeMo-co.
Carbon Monoxide↗