Solubilization of the iron molybdenum cofactor of Azotobacter vinelandii nitrogenase in dimethylformamide and acetonitrile

The iron molybdenum cofactor of Azotobacter vinelandii nitrogenase has been solubilized for the first time in dimethylformamide and acetonitrile. These solutions have the ability to reconstitute the inactive nitrogenase of the UW 45 mutant of A. vinelandii and exhibit an S = 3/2 EPR signal similar to that for the cofactor in N-methylformamide. Our ability to obtain solutions of FeMoco in these solvents seemingly refutes a previous hypothesis concerning the necessity of solvents with a dissociable proton for iron molybdenum cofactor solubility and should facilitate the spectroscopic characterization of this important species.     

光谱技术研究固氮酶铁硫簇的理化特性

Ｎ－甲基甲酰胺碱度是提取高质量固氮酶铁钼辅基的关键因素之一。过量的亚甲蓝能氧化并分解铁铜铺基为含双相铁硫簇和铁硫簇固氮酶铁钼辅基和在紫外可见光谱区中均无特征吸收峰,而在３２０ｎｍ处却呈弱吸收峰,棕色固氮菌固氮酶和该菌的突变菌侏ＵＷ４５固氮酶（缺铁钼辅基）中的非含钼的铁硫簇在紫外可见光谱区３２０ｎｍ和４０５ｎｍ处均含有特征吸收峰．     

Oxidation-reduction properties and complexation reactions of the iron-molybdenum cofactor of nitrogenase

The interactions of the iron-molybdenum cofactor, FeMoco, isolated from acid-treated Azotobacter vinelandii molybdenum-iron protein (Av1) with EDTA and thiophenol in N-methylformamide solution have been reinvestigated. Our studies show that EDTA alone is sufficient to eliminate the EPR signal of dithionite-reduced FeMoco. Neither light/5-deazaflavin nor carbon monoxide are required, which implies that this EPR-silent form of FeMoco does not correspond to the EPR-silent, substrate-reducing state of Av1. As EDTA-treated FeMoco does not regain EPR activity on addition of sodium dithionite or thiophenol, it is apparently distinct from the EPR-silent form of either dye-oxidized FeMoco or dye-oxidized Av1. Thiophenol sharpens the EPR signal of dithionite-reduced FeMoco and shifts the g = 3.3 feature to g = 3.6. This shift is complete at 1:1 ratio of thiophenol/Mo atom, while the EDTA effect requires about 40 molecules/Mo atom. Thiophenol and EDTA probably affect different sites of FeMoco. The binding of either reactant does not affect the activity of FeMoco as measured by its ability to reconstitute extracts of A. vinelandii mutant UW45.     

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.     

Apodinitrogenase: purification, association with a 20-kilodalton protein, and activation by the iron-molybdenum cofactor in the absence of dinitrogenase reductase

The Azotobacter vinelandii mutant strain UW45 contains a mutation in the nifB gene and produces an inactive dinitrogenase protein that can be activated by the addition of purified iron-molybdenum cofactor (FeMoco). This FeMoco-deficient dinitrogenase (Apo I) has now been purified 96-fold to greater than 95% purity and is FeMoco-activatable to 2200 nmol of C2H2 reduced/(min.mg of protein). The Apo I complex was found to contain two molecules of a 20-kDa protein, in addition to the alpha 2 beta 2 tetramer found for isolated holodinitrogenase (Holo I). The Apo I complex contained 15 +/- 2 mol of Fe per mole, but no Mo. While the presence of dinitrogenase reductase caused a 2-fold stimulation in the activation of the purified Apo I complex by FeMoco, this enhancement resulted from the stabilization of Apo I by dinitrogenase reductase to the denaturing effects of N-methylformamide. When the activation was performed in the absence of N-methylformamide, there was no enhancement by dinitrogenase reductase alone or by dinitrogenase reductase-Mg-ATP complex. The Apo I complex is more sensitive to O2 than Holo I, with a half-life in air of 6 min; however, the addition of dithiothreitol to Apo I during the exposure to air (or after exposure) resulted in a half-life very similar to that seen for Holo I. This suggests that sulfhydryl(s) is (are) important for the FeMoco-activation reaction.     

Purification and spectroscopic characteristics in N-methylformamide of the Azotobacter vinelandii Fe-Mo cofactor

The iron-molybdenum cofactor from Azotobacter vinelandii can be removed from significant amounts of extraneous iron and other contaminants using anaerobic gel filtration. Electronic absorption spectra of the so-purified FeMoco along with analysis of the so-called 'easily complexed' iron are suggestive that FeMoco occupies at least two different states in N-methylformamide solution. Batch-related differences in spectral characteristics of independently isolated FeMoco samples are demonstrated. Non-cofactor iron, found in unpurified FeMoco, may affect the interpretation of ligand binding and other experiments probing FeMoco structure and reactivity. Oxidized FeMoco is shown to be clearly discernable from the semi-reduced species by means of electronic spectroscopy, and this method now forms a convenient analytical tool for study of the chemistry and electronic structure of FeMoco.     

Elicitation of thiomolybdates from the iron-molybdenum cofactor of nitrogenase. Comparison with synthetic Fe-Mo-S complexes

Aerial oxidation of the iron-molybdenum cofactor (FeMoco) of Azotobacter vinelandii nitrogenase has been shown to yield either the tetrathiomolybdate ion ([MoS4]2-) or the oxotrithiomolybdate ion ([MoOS3]2-), depending on the reaction conditions. Thus, when N-methylformamide (NMF) solutions of FeMoco either were titrated with measured aliquots of air or were diluted with air-saturated NMF, [MoOS3]2- was found to be the predominant product while dilution of NMF solutions of FeMoco with air-saturated methanol produced [MoS4]2- almost exclusively. Similar aerial oxidation of solutions of chemically synthesized Fe-Mo-S clusters showed that significant information about the molybdenum environment in these species could be deduced from the nature of the elicited thiomolybdates. The differences in decomposition products as a function of solvent are postulated to be due to the loss through precipitation of the reducing agent sodium dithionite on addition of methanol but not NMF. These overall decomposition results are discussed in the context of recent X-ray absorption spectroscopic data which suggest the presence of an 'MoS3' core in FeMoco. A possible mechanism whereby [MoS4]2- might be rapidly formed from this core is presented.     

Determination of ligand binding constants for the iron-molybdenum cofactor of nitrogenase: monomers, multimers, and cooperative behavior

Equilibrium titrations in N-methylformamide (NMF) of G-25 gel filtered (ox)-state FeMo cofactor [FeMoco(ox)] from Azotobacter vinelandii nitrogenase were carried out using sodium ethanethiolate and followed using UV/Vis absorption spectroscopy. For Fe-Moco(ox), a non-linear least squares (NLLSQ) fit to the data indicated a strong equilibrium thiolate-binding step with Keq = 1.3+/-0.2x10(6) M(-1). With 245 molar excess imidazole, cooperative binding of three ethanethiolates was observed. The best NLLSQ fit gave Keq=2.0+/-0.1x10(5) M(-2) and a Hill coefficient n=2.0+/-0.3. A Scatchard plot of these data was concave upward, indicating positive cooperativity. The fit to previously published data involving benzenethiol titration of the one-electron reduced (semi-reduced) cofactor, FeMoco(sr), as followed by EPR required a model that included both a sub-stoichiometric ratio of thiol to FeMoco(sr) and about five cooperative ligand binding sites. These constraints were met by modeling FeMoco(sr) as an aggregate, with fewer thiol binding sites than FeMoco(sr) units. The best fit model was that of FeMoco(sr) as a dodecamer with five cooperative benzenethiol binding sites, yielding a thiol binding constant of 3.32+/-0.09x10(4) M(-4.8) and a Hill coefficient n=4.8+/-0.6. The results of all the other published ligand titrations of FeMoco(sr) were similarly analyzed successfully in terms of equilibrium models that include both cooperative ligand binding and dimer-level aggregation. A possible structural model for FeMoco aggregation in NMF solution is proposed.     

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.     

M?ssbauer spectroscopy applied to the oxidized and semi-reduced states of the iron-molybdenum cofactor of nitrogenase

M?ssbauer parameters at 125K for both the oxidized and semi-reduced states of FeMoco isolated from the MoFe protein of Azotobacter vinelandii nitrogenase of delta/Fe = 0.32 and 0.37 mm/s and delta Eq = 0.84 and 0.71 mm/s, respectively, are reported. FeMoco(ox) fits the Debye model perfectly from 4.2-125K and has a S = 0 ground state. FeMoco(ox) apparently contains 10-20% FeMoco(s-r) and vice versa, possibly as a result of the spontaneous oxidation phenomenon. Quantitation of the spectra indicates a Fe:Mo ratio of 5 +/- 1:1 and the similar quadrupole splittings and isomer shifts suggest a similar environment for all iron atoms.     

Nitrogenase X: M?ssbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP. Nature of the iron centers.

Under anaerobic conditions the molybdenum-iron protein (MoFe protein) from Azotobacter vinelandii can be reversibly oxidized with thionine. Electron paramagnetic resonance studies reveal that the oxidation proceeds in two distinct phases: the MoFe protein can be oxidized by four electrons without loss of the EPR signal from the S = 3/2 cofactor centers. A second oxidation step, involving two electrons, leads to the disappearance of the cofactor EPR signal. In order to correlate the events during the thionine titration with redox reactions involving individual iron centers we have studied the MoFe proteins from A vinelandii and Clostridium pasteurianum with M?ssbauer spectroscopy. Spectra were taken in the temperature range from 1.5 K to 200 K in applied magnetic fields of up to 54 kG. Analysis of the M?ssbauer data allows us to draw three major conclusions: (1) the holoprotein contains 30 +/- 2 iron atoms. (2) Most probably, 12 iron atoms belong to two, apparently identical, iron clusters (labeled M) which we have shown previously to be structural components of the iron and molybdenum containing cofactor of nitrogenase. The M-centers can be stabilized in three distinct oxidation states, MOXe- in equilibrium MNe- in equilibrium MR. The diamagnetic (S = 0) state MOX is attained by oxidation of the native state MN with either thionine or oxygen. MR is observed under nitrogen fixing conditions. (3) The data strongly suggest that 16 iron atoms are associated with four iron centers which we propose to call P-clusters. Each P-cluster contains four spin-coupled iron atoms. In the native protein the P-clusters are in the diamagnetic state PN, yielding the M?ssbauer signature which we have labeled previously 'components D and Fe2+'. Three irons of the D-type and one iron of the Fe2+-type appear to comprise a P-cluster. A one-electron oxidation yields the paramagnetic state POX. Although the state POX is characterized by half-integral electronic spin a peculiar combination of zero-field splitting parameters and spin relaxation renders this state EPR-silent. Spectroscopically, the P-clusters are novel structures; there is, however, evidence that they are closely related to familiar 4Fe-4S centers.     

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)     

The Relationship Between the Metal Clusters and the Conformation of Molybdenum-Iron Protein from Azotobacter vinelandii

The ultraviolet CD spectrum of nitrogenase MoFe protein from Azotobacter vinelandii had a negative trough with double peaks at 208 nm and 222 nm, respectively, and the shape of the trough was similar to those of other proteins with a-helix structure. After treatment with o-phenanthroline under an aerobic or anaerobic condition, the height of the peak at 222 nm (h222 nm) decreased with the decrease of the C2H2-reduction activity, Fe content and CD spectra at both 450 nm and 660 nm, or at 450 nm of the treated proteins. However, after reconstituting with a reconstituent solution containing Na2MoO4, Na2S, dithiothreitol and either ferric homocitrate or ferric citrate, the h222 nm Of the reconstituted proteins could be restored as well as the activity, Fe content and CD spectra at both of 450 nm and 660 nm. The results show that there is a significant relationship between the metal clusters (FeMoco and P-cluster) and the conformation of MoFe protein.     

Spectroscopic and biochemical studies on protein variants of quinaldine 4-oxidase: Role of E736 in catalysis and effects of serine ligands on the FeSI and FeSII clusters

Quinaldine 4-oxidase (Qox), which catalyzes the hydroxylation of quinaldine to 1H-4-oxoquinaldine, is a heterotrimeric (LMS)2 molybdo-iron/sulfur flavoprotein belonging to the xanthine oxidase family. Variants of Qox were generated by site-directed mutagenesis. Replacement in the large subunit at E736, which is presumed to be located close to the molybdenum, by aspartate (QoxLE736D) resulted in a marked decrease in kcat app for quinaldine, while Km app was largely unaffected. Although a minor reduction of the glutamine substituted variant QoxLE736Q by quinaldine occurred, its activity was below detection, indicating that the carboxylate group of E736 is crucial for catalysis. Replacement of cysteine ligands C40, C45, or C60 (FeSII) and of the C120 or C154 ligands to FeSI in the small subunit of Qox by serine led to decreased iron contents of the protein preparations. Substitutions C40S and C45S (Fe1 of FeSII) suppressed the characteristic FeSII EPR signals and significantly reduced catalytic activity. In QoxSC154S (Fe1 of FeSI), the g-factor components of FeSI were drastically changed. In contrast, Qox proteins with substitutions of C48 and C60 (Fe2 of FeSII), and of the C120 ligand at Fe2 of FeSI, retained considerable activity and showed less pronounced changes in their EPR parameters. Taken together, the properties of the Qox variants suggest that Fe1 of both FeSI and FeSII are the reducible iron sites, whereas the Fe2 ions remain in the ferric state. The location of the reducible iron sites of FeSI and FeSII appears to be conserved in enzymes of the xanthine oxidase family.     

Nitrogenase from Bacillus polymyxa. Purification and properties of the component proteins.

A purification procedure is described for the components of Bacillus polymyxa nitrogenase. The procedure requires the removal of interfering mucopolysaccharides before the two nitrogenase proteins can be purified by the methods used with other nitrogenase components. The highest specific activities obtained were 2750 nmol C2H4 formed . min-1 . mg-1 MoFe protein and 2521 nmol C2H4 formed . min-1 . mg-1 Fe protein. The MoFe protein has a molecular weight of 215 000 and contains 2 molybdenum atoms, 33 iron atoms and 21 atoms of acid-labile sulfur per protein molecule. The Fe protein contains 3.2 iron atoms and 3.6 acid-labile sulfur atoms per molecule of 55 500 molecular weight. Each Fe protein binds two ATP molecules. The EPR spectra are similar to those of other nitrogenase proteins. MgATP changes the EPR of the Fe protein from a rhombic to an axial-type signal.     

Activation In Vitro of Mutant MoFe Proteins from Azotobacter vinelandii by Reconstituent Solutions

nifB-MoFe protein （nifB-Av1）, AnifE MoFe protein （△nifE Av1） and AnifZ MoFe protein （△nifZ Av1） were obtained by chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated, nifE deleted and nifZ deleted mutant stains （UW45, DJ35 and DJ194） of Azotobacter vinelandii Llpmann, respectively. When complemented with nltrogenase Fe protein （Av2）, AnifZ Av1 had partial activity and both nifB-Avl and △nifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein （OP Av1） or △nifZ Av1. After being Incubated with excess O-phenanthrollne （O-phen） for 150 mln at 30 ℃ and subjected to chromatography on a Sephadex G-25 column In an Ar atmosphere, nifB- Av1C, △nifE Av1C and △nifZ Av1C were obtained, respectively. Based on a calculation of Fe atoms In the Ophen-Fe compound with ε 512nm = 11 100, lost Fe atoms of nifB-Av1, △nifE Av1 and △nifZ Av1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss, △nifZ Av1 loses Its original activity. In the presence of both MgATP and Av2, these Fe-loslng proteins, but not the original proteins untreated with O-phen, could be significantly activated by reconstltuent solution （RS） composed of dlthlothreltol, ferric homocltrate, Na2S and Na2MoO4, or K2CrO4, or KMnO4. But In the absence of MgATP or Av2, the activation did not occur, with the exception that △nifZ AvlC was partially activated, and the activity was only 17%. These findings Indicate that： （I） △nifZ Avl with half P-cluster content Is somewhat different from FeMoco-deflclent nifB-Avl and ,△nifE Av1 with respect to protein conformation either before or after treatment with O-phen; （11） full activation of these proteins with RS requires pretreatment with O-phen and the simultaneous presence of MgATP and Av2.     

New insights into structure-function relationships in nitrogenase: A 1.6 A resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein.

The X-ray crystal structure of Klebsiella pneumoniae nitrogenase component 1 (Kp1) has been determined and refined to a resolution of 1.6 A, the highest resolution reported for any nitrogenase structure. Models derived from three 1.6 A resolution X-ray data sets are described; two represent distinct oxidation states, whilst the third appears to be a mixture of both oxidized and reduced states (or perhaps an intermediate state). The structures of the protein and the iron-molybdenum cofactor (FeMoco) appear to be largely unaffected by the redox status, although the movement of Ser beta90 and a surface helix in the beta subunit may be of functional significance. By contrast, the 8Fe-7S P-cluster undergoes discrete conformational changes involving the movement of two iron atoms. Comparisons with known component 1 structures reveal subtle differences in the FeMoco environment, which could account for the lower midpoint potential of this cluster in Kp1. Furthermore, a non-proline- cis peptide bond has been identified in the alpha subunit that may have a functional role. It is within 10 A of the FeMoco and may have been overlooked in other component 1 models. Finally, metal-metal and metal-sulphur distances within the metal clusters agree well with values derived from EXAFS studies, although they are generally longer than the values reported for the closely related protein from Azotobacter vinelandii. A number of bonds between the clusters and their ligands are distinctly longer than the EXAFS values, in particular, those involving the molybdenum atom of the FeMoco.     

Characterization of Azotobacter vinelandii nifZ deletion strains. Indication of stepwise MoFe protein assembly

The nifZ gene product (NifZ) of Azotobacter vinelandii has been implicated in MoFe protein maturation. However, its exact function in this process remains largely unknown. Here, we report a detailed biochemical/biophysical characterization of His-tagged MoFe proteins purified from A. vinelandii nifZ and nifZ/nifB deletion strains DJ1182 and YM6A (Delta nifZ and Delta nifZ Delta nifB MoFe proteins, respectively). Our data from EPR, metal, activity, and stability analyses indicate that one alpha beta subunit pair of the Delta nifZ MoFe protein contains a P cluster ([8Fe-7S]) and an iron-molybdenum cofactor (FeMoco) ([Mo-7Fe-9S-X-homocitrate]), whereas the other contains a presumed P cluster precursor, possibly comprising a pair of [4Fe-4S]-like clusters, and a vacant FeMoco site. Likewise, the Delta nifZ Delta nifB MoFe protein has the same composition as the Delta nifZ MoFe protein except for the absence of FeMoco, an effect caused by the deletion of the nifB gene. These results suggest that the MoFe protein is likely assembled stepwise, i.e. one alpha beta subunit pair of the tetrameric MoFe protein is assembled prior to the other, and that NifZ might act as a chaperone in the assembly of the second alpha beta subunit pair by facilitating a conformational rearrangement that is required for the formation of the P cluster through the condensation of two [4Fe-4S]-like clusters. The possibility of NifZ exercising its effect through the Fe protein was ruled out because the Fe proteins from nifZ and nifZ/nifB deletion strains are not defective in their normal functions. However, the detailed mechanism of how NifZ carries out its exact function in MoFe protein maturation awaits further investigation.     

缺失nifE的棕色固氮菌突变种MoFe蛋白的纯化及体外激活

经DEAE纤维素、Sephacryl S-300和Q-Sepharose柱层析分离纯化,从缺失nifE的棕色固氮菌(Azotobactervinelandii Lipmann)突变种(DJ35)的无细胞粗提物中得到△nifE MoFe蛋白(△nifE Av1).SDS凝胶电泳分析表明,△nifE Av1的亚单位种类和分子量分别与棕色固氮菌野生型(OP)MoFe蛋白(Av1)的α和β亚单位相似.当与固氮酶Fe蛋白(Av2)活性互补时,△nifE Av1不具有还原质子的能力,但从OP Av1中抽提的FeMoco却可使其激活.经过量的邻菲啰啉(o-phen)厌氧处理并经Sephadex G-25柱层析分离后,便得到△nifE Av1 .在同时存在Av2和MgATP发生系统的条件下,△nifE Av1 ,而不是△nifE Av1,可为由KMnO4、高柠檬酸铁、Na2S、Na2S2O4和二硫苏糖醇组成的含Mn重组液(RS-Mn)显著激活.但在缺少MgATP或Av2的条件下,RS-Mn则不能激活△nifE Av1 .这就表明,RS-Mn对△nifE Av1 的激活需要o-phen的预先处理及同时存在Av2和MgATP的这二个条件.     

Variable-temperature,variable-field magnetic circular dichroism spectroscopic study of NifEN-bound precursor and “FeMoco”

NifEN plays a key role in the biosynthesis of the iron–molybdenum cofactor (FeMoco) of nitrogenase. A scaffold protein that hosts the conversion of a FeMoco precursor to a mature cofactor, NifEN can assume three conformations during the process of FeMoco maturation. One, designated ΔnifB NifEN, contains only two permanent [Fe₄S₄]-like clusters. The second, designated NifEN^Precursor, contains the permanent clusters and a precursor form of FeMoco. The third, designated NifEN^“FeMoco”, contains the permanent [Fe₄S₄]-like clusters and a fully complemented, “FeMoco”-like structure. Here, we report a variable-temperature, variable-field magnetic circular dichroism spectroscopic investigation of the electronic structure of the metal clusters in the three forms of dithionite-reduced NifEN. Our data indicate that the permanent [Fe₄S₄]-like clusters are structurally and electronically conserved in all three NifEN species and exhibit spectral features of classic [Fe₄S₄]⁺ clusters; however, they are present in a mixed spin state with a small contribution from the S > ½ spin state. Our results also suggest that both the precursor and “FeMoco” have a conserved Fe/S electronic structure that is similar to the electronic structure of FeMoco in the MoFe protein, and that the “FeMoco” in NifEN^“FeMoco” exists, predominantly, in an S = 3/2 spin state with spectral parameters identical to those of FeMoco in the MoFe protein. These observations provide strong support to the outcome of our previous EPR and X-ray absorption spectroscopy/extended X-ray absorption fine structure analysis of the three NifEN species while providing significant new insights into the unique electronic properties of the precursor and “FeMoco” in NifEN.