An atomic-level mechanism for molybdenum nitrogenase. Part 2. Proton reduction,inhibition of dinitrogen reduction by dihydrogen,and the HD formation reaction

Quantum calculations have been used to examine the energetics of the reactions of diazene and isodiazene with H(2) and the properties of the Fe and Mo sites of the nitrogenase iron-molybdenum cofactor with respect to the binding of H and H(2). The results have been used to extend the model for N(2) reduction by nitrogenase given in the preceding paper to describe the formation of HD from D(2). The proposed mechanism for HD formation invokes a combination of two well-established chemical reactions, namely, competitive protonation of metal N(2) species at either the metal or at N(2), followed by scrambling of D(2) at a metal hydride. The model is evaluated against the available biochemical data for the nitrogenase HD formation reaction and extended to account for H(2) inhibition of N(2) reduction and the reduction of H(+) in the absence of other substrates.     

An atomic level model for the interactions of molybdenum nitrogenase with carbon monoxide, acetylene, and ethylene

A combination of density functional theory and molecular mechanics calculations has been used to study the possible interactions of CO, C(2)H(2), and C(2)H(4) with the central Fe and terminal Mo sites of the iron-molybdenum cofactor of nitrogenase. The most favorable binding mode for CO on the central section of the FeMoco appears to be end-on to a single Fe and results in a change from high to low spin for the ligating Fe atom. If a coordination site for CO is available on the Mo, this becomes the preferred CO binding site. Calculated nu(CO) infrared frequencies are compared with the experimental values given in the literature. C(2)H(2) binds weakly in a side-on orientation to a single Fe site; addition of a single H(+)/e(-) couple to the substrate results in spontaneous migration of the resulting -CH=CH(2) group from Fe to a central S atom of the cofactor. Further reduction liberates C(2)H(4) or alternatively can give an S=CHCH(3) intermediate, which then goes on to produce C(2)H(6). A model for C(2)H(2) reduction by nitrogenase is proposed, based on the results of the calculations and the extensive literature on this process.     

Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase.

A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.     

Molybdenum independence of nitrogenase component synthesis in the non-heterocystous cyanobacterium Plectonema.

The cyanobacterium Plectonema boryanum (IU 594-UTEX 594) fixes N2 only in the absence of combined N and of O2. We induced nitrogenase by transfer to anaerobic N-free medium and studied the effect of Mo starvation on nitrogenase activity and synthesis. Activity was first detected within 3 h after transfer by the acetylene reduction assay in controls, increasing for at least 25 h. Cells grown on nitrate and Mo and then transferred to N-free, Mo-free medium produced 8% of the control nitrogenase activity. Addition of W to the Mo-free medium reduced the activity to 0.5%. Under both Mo starvation conditions, nitrogenase protein components were synthesized. Component II of the cyanobacterial enzyme was detected by in vitro complementation with Mo-containing component I from Klebsiella pneumoniae or Azotobacter vinelandii but not Clostridium pasteurianum. Component I activity was restored by addition of Mo to cultures in which new enzyme synthesis was blocked by chloramphenicol. Acidified extracts of Plectonema induced in Mo-containing medium contained the Fe-Mo cofactor required to activate extracts of the Azotobacter mutant UW45 in vitro, but they did not activate extracts of Mo-starved Plectonema. Analysis of 35SO4(2-)-labeled proteins by polyacrylamide gel electrophoresis suggested that Mo is required for the conversion of a high-molecular-weight precursor to component I in Plectonema.     

Nucleotide and divalent cation specificity of in vitro iron-molybdenum cofactor synthesis.

The nucleotide and divalent cation requirements of the in vitro iron-molybdenum cofactor (FeMo-co) synthesis system have been compared with those of substrate reduction by nitrogenase. The FeMo-co synthesis system specifically requires ATP, whereas both 1,N6-etheno-ATP and 2'-deoxy-ATP function in place of ATP in substrate reduction (M. F. Weston, S. Kotake, and L. C. Davis, Arch. Biochem. Biophys. 225:809-817, 1983). Mn2+, Ca2+, and Fe2+ substitute for Mg2+ to various extents in in vitro FeMo-co synthesis, whereas Ca2+ is ineffective in substrate reduction by nitrogenase. The observed differences in the nucleotide and divalent cation specificities suggest a role(s) for the nucleotide and divalent cation in in vitro FeMo-co synthesis that is distinct from their role(s) in substrate reduction.     

A new method for extraction of iron-molybdenum cofactor (FeMoco) from nitrogenase adsorbed to DEAE-cellulose. 1. Effects of anions, cations, and preextraction treatments

A convenient and rapid method of obtaining the cofactor of nitrogenase (FeMoco) with a low and apparently limiting Fe/Mo ratio has been developed. FeMoco can be extracted from the MoFe protein bound to DEAE-cellulose. The cofactor is eluted in either N-methylformamide (NMF), N,N-dimethylformamide (DMF), or mixtures of these solvents by use of salts such as Et4NBr,Bu4NBr,Ph4PCl, and Ph4AsCl. The method is simple, is rapid (45 min), yields concentrated cofactor, and, unlike the original method [Shah, V. K., & Brill, W. J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3249-3253] which requires anaerobic centrifugation, is easily scaled up. Furthermore, it gives yields of cofactor in excess of 70%. Its disadvantages are a high Fe:Mo ratio when DMF is the extracting solvent and a high salt concentration in the resultant FeMoco solution. These disadvantages are easily overcome by removing excess Fe by pretreating the cofactor with bipyridyl while still on the column. This gives Fe:Mo ratios of (6 +/- 1):1 (11 trials) with specific activities ranging from 170 to 220 nmol of C2H4/[min.(nmol of Mo)]. Chromatography on Sephadex LH-20 removes ca. 99% of the excess salt. The adsorption of MoFe protein to DEAE-cellulose seems to facilitate denaturation by organic solvents so that pretreatment of the protein with acid, used in earlier methods, is unnecessary. There is an apparent dependence on the charge density of the anion employed for elution of FeMoco bound to DEAE-cellulose, such that Cl- greater than Br- much greater than I-, PF6- is the order of effectiveness of the Bu4N+ salts of these anions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analogs for the reduction of certain nitrogenase substrates. Are multiple sites within the Fe/Mo/S active center involved in the 6e– reduction of N2?

 Reactivity studies of clusters that contain the MFe₃S₄ cores (M = Mo, V) with catecholate, multicarboxylate (or DMF) ligands coordinated to the Mo (or V) atoms, and Cl ligands coordinated to the Fe atoms have been carried out. These studies show the M/Fe/S single cubane clusters to be effective catalysts in the reduction of nitrogenase substrates such as hydrazine, acetylene and protons to give ammonia, ethylene and dihydrogen respectively. The same molecules do not activate or catalyze the reduction of dinitrogen. The results indicate that the observed catalyses are occurring at the Mo (V) sites by a process that, in the case of hydrazine, involves substrate protonation prior to reduction. The facile catalytic reduction of hydrazine by clusters that contain coordinatively saturated polycarboxylate-bound Mo atoms is rationalized in terms of a possible protonation/proton delivery function of the coordinated polycarboxylate ligands. The reactivity characteristics of the M/Fe/S clusters (structurally quite similar to the nitrogenase cofactor) have led to the suggestion that the Mo (V) atoms may be involved in the reduction of hydrazine in the later stages of dinitrogen reduction. Received and accepted: 21 August 1996     

Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane.     

Characteristics of N2 fixation in Mo-limited batch and continuous cultures of Azotobacter vinelandii.

Steady-state chemostat cultures of Azotobacter vinelandii were established in a simple defined medium that had been chemically purified to minimize Mo and that contained no utilizable combined N source. Growth was dependent on N2 fixation, the limiting nutrient being the Mo contaminating the system. The Mo content of the organisms was at least 100-fold lower than that of Mo-sufficient cultures, and they lacked the characteristic g = 3.7 e.p.r. feature of the MoFe-protein of nitrogenase. A characteristic of nitrogenase activity in vivo in Mo-limited populations was a disproportionately low activity for acetylene reduction, which was 0.3 to 0.1 of that expected from the rate of N2 reduction. Acetylene was also a poor substrate in comparison with protons as a substrate for nitrogenase, and did not markedly inhibit H2 evolution, in contrast with Mo-sufficient populations. In batch cultures in similar medium or 'spent' chemostat medium inoculated with Mo-limited organisms, the addition of Mo elicited a biphasic increased growth response at concentrations as low as 2.5 nM, provided that sufficient Fe was supplied. In this system V did not substitute for Mo, and Mo-deficient cultures ceased growth at a 25-fold lower population density compared with cultures supplemented with Mo. Nitrogenase component proteins could not be unequivocally detected by visual inspection of fractionated crude extracts of Mo-limited organisms. 35SO42-pulse-labelling studies also showed that the rate of synthesis of the MoFe-protein component of nitrogenase was too low to be quantified. However, the Fe-protein of nitrogenase was apparently synthesized at high rates. The discussion includes an evaluation of the possibility that A. vinelandii possesses an Mo-independent N2-fixation system.     

Substrate interactions with nitrogenase: Fe versus Mo

Biological nitrogen reduction is catalyzed by a complex two-component metalloenzyme called nitrogenase. For the Mo-dependent enzyme, the site of substrate reduction is provided by a [7Fe-9S-Mo-X-homocitrate] metallocluster, where X is proposed to be an N atom. Recent progress with organometallic model compounds, theoretical calculations, and biochemical, kinetic, and biophysical studies on nitrogenase has led to the formulation of two opposing models of where N(2) or alternative substrates might bind during catalysis. One model involves substrate binding to the Mo atom, whereas the other model involves the participation of one or more Fe atoms located in the central region of the metallocluster. Recently gathered evidence that has provided the basis for both models is summarized, and a perspective on future research in resolving this fundamental mechanistic question is presented.     

The Fe-only nitrogenase from Rhodobacter capsulatus: identification of the cofactor, an unusual, high-nuclearity iron-sulfur cluster, by Fe K-edge EXAFS and 57Fe Mössbauer spectroscopy

Samples of the dithionite-reduced FeFe protein (the dinitrogenase component of the Fe-only nitrogenase) from Rhodobacter capsulatus have been investigated by 57Fe M?ssbauer spectroscopy and by Fe and Zn EXAFS as well as XANES spectroscopy. The analyses were performed on the basis of data known for the FeMo cofactor and the P cluster of Mo nitrogenases. The prominent Fourier transform peaks of the Fe K-edge spectrum are assigned to Fe-S and Fe-Fe interactions at distances of 2.29 A and 2.63 A, respectively. A significant contribution to the Fe EXAFS must be assigned to an Fe backscatterer shell at 3.68 A, which is an unprecedented feature of the trigonal prismatic arrangement of iron atoms found in the FeMo cofactor of nitrogenase MoFe protein crystal structures. Additional Fe...Fe interactions at 2.92 A and 4.05 A clearly indicate that the principal geometry of the P cluster is also conserved. M?ssbauer spectra of 57Fe-enriched FeFe protein preparations were recorded at 77 K (20 mT) and 4.2 K (20 mT, 6.2 T), whereby the 4.2 K high-field spectrum clearly demonstrates that the cofactor of the Fe-only nitrogenase (FeFe cofactor) is diamagnetic in the dithionite-reduced ("as isolated") state. The evaluation of the 77 K spectrum is in agreement with the assumption that this cofactor contains eight Fe atoms. In the literature, several genetic and biochemical lines of evidence are presented pointing to a significant structural similarity of the FeFe, the FeMo and and the FeV cofactors. The data reported here provide the first spectroscopic evidence for a structural homology of the FeFe cofactor to the heterometal-containing cofactors, thus substantiating that the FeFe cofactor is the largest iron-sulfur cluster so far found in nature.     

Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life's key biogeochemical innovations. The three forms of nitrogenase differ in their metal dependence, each binding either a FeMo‐, FeV‐, or FeFe‐cofactor where the reduction of dinitrogen takes place. The history of nitrogenase metal dependence has been of particular interest due to the possible implication that ancient marine metal availabilities have significantly constrained nitrogenase evolution over geologic time. Here, we reconstructed the evolutionary history of nitrogenases, and combined phylogenetic reconstruction, ancestral sequence inference, and structural homology modeling to evaluate the potential metal dependence of ancient nitrogenases. We find that active‐site sequence features can reliably distinguish extant Mo‐nitrogenases from V‐ and Fe‐nitrogenases and that inferred ancestral sequences at the deepest nodes of the phylogeny suggest these ancient proteins most resemble modern Mo‐nitrogenases. Taxa representing early‐branching nitrogenase lineages lack one or more biosynthetic nifE and nifN genes that both contribute to the assembly of the FeMo‐cofactor in studied organisms, suggesting that early Mo‐nitrogenases may have utilized an alternate and/or simplified pathway for cofactor biosynthesis. Our results underscore the profound impacts that protein‐level innovations likely had on shaping global biogeochemical cycles throughout the Precambrian, in contrast to organism‐level innovations that characterize the Phanerozoic Eon.     

Insertion of heterometals into the NifEN-associated iron–molybdenum cofactor precursor

The cofactors of Mo-, V-, Fe-dependent nitrogenases are believed to be highly homologous in structure despite the different types of heterometals (Mo, V, and Fe) they contain. Previously, a precursor form of the FeMo cofactor (FeMoco) was captured on NifEN, a scaffold protein for FeMoco biosynthesis. This all-Fe precursor closely resembles the Fe/S core structure of the FeMoco and, therefore, could reasonably serve as a precursor for all nitrogenase cofactors. Here, we report the heterologous incorporation of V and Fe into the NifEN-associated FeMoco precursor. EPR and activity analyses indicate that V and Fe can be inserted at much reduced efficiencies compared with Mo, and incorporation of both V and Fe is enhanced in the presence of homocitrate. Further, native polyacrylamide gel electrophoresis experiments suggest that NifEN undergoes a significant conformational rearrangement upon metal insertion, which allows the subsequent NifEN–MoFe protein interactions and the transfer of the cofactor between the two proteins. The combined outcome of these in vitro studies leads to the proposal of a selective mechanism that is utilized in vivo to maintain the specificity of heterometals in nitrogenase cofactors, which is likely accomplished through the redox regulation of metal mobilization by different Fe proteins (encoded by nifH, vnfH, and anfH, respectively), as well as the differential interactions between these Fe proteins and their respective scaffold proteins (NifEN and VnfEN) in the Mo-, V-, and Fe-dependent nitrogenase systems.     

A facile method of isolation for the iron molybdenum cofactor of nitrogenase and bacterial ferritin from extracts of azotobacter vinelandii

An alternative method has been developed for the isolation of both the iron molybdenum cofactor of nitrogenase (FeMoco), a small molecular weight FeMoS cluster which is the putative nitrogen- reducing site of the enzyme, and bacterioferritin, an iron storage protein similar to other ferritins, but containing heme prosthetic groups. Previously the isolation of these two species, the characterization of which is of significant current interest, has been dependent on the purification of the nitrogenase enzyme from Azotobacter vinelandii. Out new procedure eliminates the use of the anaerobic column chromatography necessary to obtain pure nitrogenase components, involving instead the heat and RNAase/ DNAase treatment of crude extracts of ruptured cells followed by sedimentation (150000 × g for 18 h) of both the 'nitrogenase complex' and bacterioferritin. The redissolved pellet from this centrifugation yields the pure crystalline bacterioferritin on addition of Mg²⁺. and cooling, the iron content of the protein being higher by this method than in previous reports. Likewise, denaturation by acid/base treatment of this protein mixture yields a precipitate which can be extracted with either N-methylformamide or N,N-dimethylformamide containing dithionite ion to yield solutions of FeMoco, as evidenced by UW 45 reconstitution and EPR spectral criteria. Unfortunately, preparations of FeMoco obtained by this method have a variable, but consistently low, Fe/Mo ratio and additional visible spectral features, indicating that they are significantly less pure than that those generated from purified nitrogenase. The aqueous supernatant from the denaturation also yields bacterioferritin, but with a lower iron content than that from the direct crystallization method.     

Metal limitation of cyanobacterial N2 fixation and implications for the Precambrian nitrogen cycle

Nitrogen fixation is a critical part of the global nitrogen cycle, replacing biologically available reduced nitrogen lost by denitrification. The redox‐sensitive trace metals Fe and Mo are key components of the primary nitrogenase enzyme used by cyanobacteria (and other prokaryotes) to fix atmospheric N₂ into bioessential compounds. Progressive oxygenation of the Earth's atmosphere has forced changes in the redox state of the oceans through geologic time, from anoxic Fe‐enriched waters in the Archean to partially sulfidic deep waters by the mid‐Proterozoic. This development of ocean redox chemistry during the Precambrian led to fluctuations in Fe and Mo availability that could have significantly impacted the ability of prokaryotes to fix nitrogen. It has been suggested that metal limitation of nitrogen fixation and nitrate assimilation, along with increased rates of denitrification, could have resulted in globally reduced rates of primary production and nitrogen‐starved oceans through much of the Proterozoic. To test the first part of this hypothesis, we grew N₂‐fixing cyanobacteria in cultures with metal concentrations reflecting an anoxic Archean ocean (high Fe, low Mo), a sulfidic Proterozoic ocean (low Fe, moderate Mo), and an oxic Phanerozoic ocean (low Fe, high Mo). We measured low rates of cellular N₂ fixation under [Fe] and [Mo] estimated for the Archean ocean. With decreased [Fe] and higher [Mo] representing sulfidic Proterozoic conditions, N₂ fixation, growth, and biomass C:N were similar to those observed with metal concentrations of the fully oxygenated oceans that likely developed in the Phanerozoic. Our results raise the possibility that an initial rise in atmospheric oxygen could actually have enhanced nitrogen fixation rates to near modern marine levels, providing that phosphate was available and rising O₂ levels did not markedly inhibit nitrogenase activity.     

Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium.

We have constructed a strain of Azotobacter vinelandii which has deletions in the genes for both the molybdenum (Mo) and vanadium (V) nitrogenases. This strain fixed nitrogen in medium that did not contain Mo or V. Growth and nitrogenase activity were inhibited by Mo and V. In highly purified medium, growth was limited by iron. Addition of other metals (Co, Cr, Cu, Mn, Ni, Re, Ti, W, and Zn) did not stimulate growth. Like the V-nitrogenase, the nitrogenase synthesized by the double deletion strain reduced acetylene to both ethylene and ethane (C2H6/C2H4 ratio, 0.046). There was an approximately 10-fold increase in ethane production when Mo was added to the deletion strain grown in medium lacking Mo and V. This change in reactivity may be due to the incorporation of an Mo-containing cofactor into the nitrogenase synthesized by the double-deletion strain. A strain synthesizing the V-nitrogenase did not show a similar increase in ethane production. The growth characteristics of the double-deletion strain, together with the metal composition reported for a nitrogenase isolated from a tungstate-tolerant strain lacking genes for the molydenum enzyme grown in the absence of Mo and V (J. R. Chisnell, R. Premakumar, and P. E. Bishop, J. Bacteriol. 170:27-33, 1988) show that A. vinelandii can synthesize a nitrogenase which lacks both Mo and V. Reduction of dinitrogen by nitrogenase can therefore occur at a center lacking both these metals.     

Purification and Characterization of Cr-containing Nitrogenase Component I

A mutant UW3, which is unable to fix N2 in the presence of Mo (Nif-) but can undergo phenotypic reversal to Nif+ under Mo deficient conditions, was able to grow in Cr containing but Mo and NH3 deficient medium. A partly purified nitrogenase component Ⅰ protein obtained from UW3 grown on the Cr containing medium was shown to contain Fe and Cr (atom ratio of Fe to Cr and Mo to Cr: 11.60 and 0.41) and to have 70% of the C2H2 and H+ reduction activity of MoFe protein from the wild type strain of Azotobacter vinelandii Lipmann. The Cr containing protein was different in subunit composition from that of MnFe protein purified from the mutant strain grown in the presence of Mn, but similar to that of MoFe protein, that is, it was a tetramer composed of two differentsubunits (α2β2). The preliminary results indicated that the Cr containing protein might be a nitrogenase component Ⅰ protein.     

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation.

A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.