Purification and Characteristics of Mn-containing Nitrogenase Component

A mutant UW3, which is unable to fix N2 in the presence of Mo (Nif-) but undergo phenotypic reversal to Nif+ under Mo deficiency, was able to grow in Mo- and NH3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C2H2- and H+-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰprotein.     

固氮酶铬铁蛋白的晶体生长

在合适的结晶条件下,从含Cr无氨培养基中生长的固氮菌(Azotobacter vinelandii Lipmann)突变种UW3中纯化出的CrFe蛋白可从溶液中析出深棕色斜四棱柱晶体,晶体最大的两条对角线长度分别可达0.25 mm和0.12 mm.PEG 8000、MgCl2、NaCl、Tris 和Hepes 缓冲液的浓度及结晶方法等对该蛋白的出晶率、晶核数目、晶体大小和质量都有明显影响.CrFe蛋白结晶所需的上述化合物的最适浓度与在Mn中生长的固氮菌突变种UW3的MnFe蛋白和缺失nifZ固氮菌突变种的ΔnifZ MoFe蛋白结晶所需的最适浓度有所不同.结果表明,该蛋白晶体可能为CrFe蛋白的晶体.     

固氮酶CrFe蛋白和MnFe蛋白的空间晶体生长

从分别牛长于含Mn和Cr培养基中的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3分离纯化出MnFe和CrFe蛋白。为适应包括固氮酶在内的氧敏感蛋白的空间晶体生长的要求,应用简易而适用的厌氧加样装置代替固氮酶实验室所用的笨重厌氧箱(dry box),在地面进行厌氧加样。在充满氮气的简便有机玻璃箱内厌氧加样的所有样品中,分别用液／液扩散法和汽相扩散的坐滴法都可在一周内使MnFe和CrFe蛋白在宇宙飞船上从溶液中结晶出来。在所用的数种蛋白沉淀剂中,飞船上形成的所有晶体都为单品,而地面上在多数沉淀剂中都生成大量挛晶。在相同沉淀剂中用液／液扩散法,飞船上生成CrFe蛋白的最大晶体比地面生成的最大晶体大1倍。而在相同沉淀剂中用汽相扩散的坐滴法,飞船上生成的MnFe蛋白最大晶体却没有地面生成的最大晶体大。这种差异也许是由不同结晶方法而不是不同蛋白所引起的。     

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．     

Characteristics and Crystallization of Nitrogenase MoFe Protein from a nif Z Deleted Strain of Azotobacter vinelandii

△nifZ MoFe protein purified from a nifZ deleted strain of Azotobacter vinelandii (DJ194) was shown to be pure by SDS-Polyacrylamide gel electrophoresis. The protein contained 1.5 Mo atoms and 15.9 Fe atoms per molecule, the ratio of Fe to Mo was lower than that of the MoFe protein purified from the wild type strain of A. vinelandii; and Call2, H+ -reduction activity and their ratio (C2H4/H2 (Ar)) were 16.6%, 21.7% and 77.2% of those of the wild type MoFe protein, respectively. Under a somewhat different condition from that for the crystallization of the wild type MoFe protein dark brown rhombohedron crystals of △nifZ MoFe protein were obtained. It indicated that the deletion of the △nif Z resulted in the decrease of number or change in the structure of P-cluster in the mutant MoFe protein, which caused the significant structured and function of change of the protein.     

Growth of the Crystals of Nitrogenase MnFe Protein (in English)

Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing but Mo- and NH3-free medium. The possibility of crystallization, and number,size and quality of crystals were obviously dependent on concentrations of NaCl,MgCl2, PEG 8000,Tris and Hepes buffer and on methods for crystallization. PEG concentration affected on the shape of the crystals.The optimal concentrations of the chemicals for crystallization of MnFe protein were slightly different from those for crystallization of ΔnifZ MoFe protein from a nifZ deleted strain of Azotobacter vinelandii .　SDS-PAGE showed that the protein from the dissolved crystals was almost the same as MnFe protein before crystallization, indicating that the crystal was formed from MnFe protein.     

Purification and properties of the nitrogenase of Azospirillum amazonense.

The nitrogenase of the free-living, microaerobic, N2-fixing bacterium Azospirillum amazonense (strain Y1) was purified by chromatography on DEAE-52 cellulose, by heat treatment, and by preparative polyacrylamide gel electrophoresis. The specific nitrogenase activities were 2,400 nmol of C2H4 formed per min per mg of protein for dinitrogenase (MoFe protein) and 1,800 nmol of C2H4 formed per min per mg of protein for dinitrogenase reductase (Fe protein). The MoFe protein was composed of a minimum of 1,852 amino acid residues, had an isoelectric point of 5.2, and contained 2 atoms of Mo, 24 atoms of Fe, and 28 atoms of acid-labile sulfide per molecule. The Fe protein had 624 amino acid residues and an isoelectric point of 4.6 and contained four atoms of Fe and six atoms of acid-labile sulfide per molecule. The purified MoFe protein showed two subunits with molecular weights of 55,000 and 50,000. The purified Fe protein revealed two polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular weights of 35,000 and 31,000. The two Fe protein polypeptides were demonstrated with immunological techniques in the purified, highly active enzyme as well as in extracts. Also, Azotobacter vinelandii Fe protein showed two closely migrating polypeptides that migrated differently from the Fe protein polypeptides of Azospirillum brasilense or Rhodospirillum rubrum. The nitrogenase activity of Azospirillum amazonense Y1 was independent of Mn2+, and the addition of activating enzyme had no effect. No activating enzyme could be found in Azospirillum amazonense. Obviously, the nitrogenase system of Azospirillum amazonense Y1 is different from that of Azospirillum brasilense Sp7 and resembles the Azotobacter system.     

Structural Requirement for Clusters to be Reconstituted with the FeMoCo-Deficient Molybdenum-Iron Protein

By incubating the reduced MoFe protein from Azotobacter vinelandii with O-phenanthroline under air and chromatographying the incubated solution on Sephadex G-25 column, inactive MoFe protein could be obtained. Its acetylene-reduction activity was remarkably recovered not only by incubation with the reconstituent solution composed of KMnO4, ferric homoeitrate, Na2S and dithiothreitol, but also with a mixture of 4Fe : 4S clusters and another cluster which had two structure units of 1Mo : 3Fe : 4S-bridged by three -OCH3 at the Mo atoms. Neither the reconstituent solution nor the mixture could reactivate apo-MoFe proteins from the mutants deleting nile and nifH genes and from the mutant UW45, which could be reactivated by the FeMoeo extracted from the MoFe protein. The results indicated that the FeMoeo-defieient MoFe proteins from these mutants seemed to be reconstituted only by the clusters which were probably structures only similar to FeMoeo. The partially metalloeluster-deficient MoFe protein could be reconstituted by the clusters with a certain kind of structure and composition; and was changed into different nitrogenase proteins with the ability to fix nitrogen.     

Properties of the nitrogenase system from a photosynthetic bacterium, Rhodospirillum rubrum.

Soluble nitrogenase from Rhodospirillum rubrum has been isolated and separated into its two components, the MoFe protein and the Fe protein. The MoFe protein has been purified to near homogeneity and has a molecular weight or 215 000. It contains two Mo, 25--30 Fe and 19--22 acid-labile sulphide and consists of four subunits, Mw 56 000. The Fe protein has a molecular weight 65 000. It contains approximately four Fe and four acid-labile sulphide and consists of two subunits, Mw 31 500. The highest specific activities for the purified components are 920 and 1260 nmol ethylene produced per min per mg protein, respectively. The purified components require the membrane component for activity (Nordlund, S., Eriksson, U. and Baltscheffsky, H. (1977) Biochim. Biophys. Acta 462, 187--195). Titration of the MoFe protein with the Fe protein shows saturation and excess MoFe protein over Fe protein is inhibitory. Addition of Fe2+ or Mn2+ to the reaction mixture increases the activity apparently through interaction with the membrane component.     

Activation of Inactive Nitrogenase by Acid-Treated Component I

When Azotobacter vinelandii was derepressed for nitrogenase synthesis in a N-free medium containing tungstate instead of molybdate, an inactive component I was synthesized. Although this inactive component I could be activated in vivo upon addition of molybdate to the medium, it could not be activated in vitro when molybdate was added to the extracts. Activation occurred, however, when an acid-treated component I was added to extracts of cells derepressed in medium containing tungstate. Acid treatment completely abolished component I activity. Mutant strains UW45 and UW10 were unable to fix N(2). Both strains synthesized normal levels of component II but produced inactive component I. Acid-treated component I activated inactive component I in extracts of mutant strain UW45 but not mutant strain UW10. This activating factor could be obtained from N(2)-fixing Klebsiella pneumoniae, Clostridium pasteurianum, and Rhodospirillum rubrum.     

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.     

缺失nifZ的棕色固氮菌突变种钼铁蛋白的特性和结晶

从棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ　ｖｉｎｅｌａｎｄｉｉ）缺失ｎｉｆＺ突变种中提纯得到的Δｎｉｆ　ＺＭｏＦｅ蛋白达到ＳＤＳ凝胶电泳纯。每个△ｎｆｉ　ＺＭｏＦｅ蛋白分子含１．５个Ｍｏ和１５．９个Ｆｅ原子,它的Ｆｅ和Ｍｏ比值低于野生型固氮菌ＭｏＦｅ蛋白的Ｆｅ和Ｍｏ比值,而它的Ｃ２Ｈ２、Ｈ＋还原活性及其比率（Ｃ２Ｈ４／Ｈ２（Ａｒ））分别为野生型ＭｏＦｅ蛋白的１６．６％、２１．７％和７７．２％。在与野生型ＭｏＦｅ蛋白结晶条件略有不同的情况下,所得的Δｎｉｆ　Ｚ　ＭｏＦｅ蛋白晶体为深棕色的斜四棱柱体晶体。表明ｎｉｆＺ的缺失可能使突变种ＭｏＦｅ蛋白中的Ｐ－ｃｌｕｓｔｅｒ或数目减少或结构发生变化,从而引起该蛋白的结构和功能发生明显改变。     

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.     

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.     

FeMo cofactor synthesis by a nifH mutant with altered MgATP reactivity.

We have characterized a Nif- mutant of Azotobacter vinelandii, designated UW91 (Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H., and Brill, W. J. (1973) Biochim. Biophys. Acta 292, 246-255). The specific Fe protein mutation giving rise to the Nif- phenotype was shown by DNA sequencing and site-directed mutagenesis to be the substitution of a conserved alanine at position 157 by a serine. The UW91 Fe protein was purified and shown to have a normal [4Fe-4S] cluster and normal MgATP binding activity. The substitution of alanine 157 by serine, however, prevents the MgATP-induced conformational change that occurs for the wild-type Fe protein, prevents MgATP hydrolysis, and prevents productive electron transfer to the MoFe protein. The UW91 Fe protein does bind to the MoFe protein to give a normal cross-linking pattern; however, it does not compete very successfully with wild-type Fe protein in an activity assay. The UW91 MoFe protein was also purified and characterized and shown to be indistinguishable from the wild-type protein. Thus, the substitution of Fe protein residue alanine 157 by serine does not change the Fe protein's ability to function in FeMo cofactor biosynthesis or insertion. This demonstrates that these events do not require the MgATP-induced conformational change, MgATP hydrolysis, or productive electron transfer to the MoFe protein.     

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 the inactive MoFe protein (NifB-Kp1) of the nitrogenase from nifB mutants of Klebsiella pneumoniae.

The inactive MoFe protein of nitrogenase, NifB-Kp1, from two distinct nifB mutants of Klebsiella pneumoniae, Kp5058 (a nifB point mutant) and UNF1718 (a nifB, nifJ double mutant) has been purified and characterized. NifB-Kp1 can be activated by reaction with the iron-molybdenum cofactor, FeMoco, extracted from active MoFe protein. NifB-Kp1 purified from either source had similar properties and was contaminated with an approximately equimolar amount of protein of mol.wt. 21 000. Like active wild-type Kp1, it was an alpha 2 beta 2 tetramer, but it was far less stable than Kp1, deteriorating rapidly at temperatures above 8 degrees C or on mild oxidation. NifB-Kp1 preparations contained 0.4-0.9 Mo and 9.0 +/- 0.9 Fe atoms . mol-1 and, when activated by FeMoco, had a specific activity of approx. 500 units . mg-1. The Mo in our preparations was not associated with the e.p.r. signal normally observed from FeMoco. All preparations exhibited a weak gav. = 1.95 e.p.r. signal which was probably not associated with activatable protein.     

Mutagenesis studies of the FeSII protein of Azotobacter vinelandii: roles of histidine and lysine residues in the protection of nitrogenase from oxygen damage

The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complex with nitrogenase during periods when nitrogenase is exposed to oxygen. One possible mechanism for its action is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSII protein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions. Herein we report the construction and characterization of five site-directed mutants of the FeSII protein (H12Q, H55Q, K14A, K15A, and the double mutant K14A/K15A) which were individually purified after being individually overexpressed in Escherichia coli. These mutant FeSII proteins maintain native-like assembly and orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization and their redox midpoint potentials, which are within 25 mV of that of the wild type protein. The abilities of the individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenase activities after adding each pure version back to extracts from an FeSII deletion strain, and then exposing the mixture to oxygen. In these assays, the H12Q mutant functioned as well as the wild type protein. However, mutation of His55, a few residues away from a cluster-liganding cysteine, results in much less efficient protection of nitrogenase. These results are consistent with pH titrations in both oxidation states, which show that His12 is insensitive to 2Fe-2S cluster oxidation state. His55's pK is weakly responsive to oxidation state, and the pK increase of 0. 16 pH unit upon 2Fe-2S cluster oxidation is indicative of ionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSII protein's affinity for nitrogenase in a redox state-dependent manner. Both K14A and K15A mutant FeSII proteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all its protective ability. The nitrogenase component proteins in an Azotobacter strain bearing the double lysine mutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild type strain under carbon substrate-limited conditions. These results indicate that the two lysines may have an important role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase component proteins.     

Regulation and order of involvement of molybdoproteins during synthesis of molybdoenzymes in Clostridium pasteurianum.

The accumulation of 99Mo (from 99MoO4(2-) into molybdenum-containing species in Clostridium pasteurianum was investigated to identify the molybdoprotein(s) involved in Mo metabolism. Mo accumulation by clostridial cells during the derepression of the nitrogenase system increased substantially beginning 1.5 h before nitrogenase activity was detected. The increase in Mo accumulation by the cells is a result of the incorporation of Mo into a high-molecular-weight molybdenum species (suspected membrane fragments), a low-molecular-weight molybdenum species, a Mo binding-storage protein, a 30-kilodalton molybdoprotein, and formate dehydrogenase. Mo incorporation into the MoFe protein was detected 1 h after the onset of metal uptake. Kinetics of Mo accumulation into the molybdoproteins during the derepression of nitrogenase suggests that Mo incorporation or uptake or both occur in the following sequence: (i) membranes and MoO4(2-), (ii) a low-molecular-weight molybdenum species, (iii) Mo binding-storage protein and a 30-kilodalton molybdoprotein, (iv) formate dehydrogenase, and (v) the MoFe protein. The intracellular level of all molybdenum components except the MoFe protein appears to be influenced by the availability of Mo. Clostridial cells grown in the presence of a limiting amount of Mo became Mo deficient as a result of growth and a MoO4(2-) supplement added to such cells rapidly accumulated within the cells to levels five times that found in steady-state nitrogen-fixing cells. The Mo accumulated by the Mo-deficient cells was rapidly incorporated into preformed demolybdoproteins in the absence of de novo protein synthesis. The increase in Mo accumulation by Mo-deficient cells was a result of an increase in all molybdoproteins except the MoFe protein.     

固氮酶CrFe蛋白和MnFe蛋白的空间晶体生长

从分别生长于含Mn和Cr培养基中的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3分离纯化出MnFo和CrFe蛋白.为适应包括固氮酶在内的氧敏感蛋白的空间晶体生长的要求,应用简易而适用的厌氧加样装置代替固氮酶实验室所用的笨重厌氧箱(dry box),在地面进行厌氧加样.在充满氮气的简便有机玻璃箱内厌氧加样的所有样品中,分别用液/液扩散法和汽相扩散的坐滴法都可在一周内使MnFe和CrFe蛋白在宇宙飞船上从溶液中结晶出来.在所用的数种蛋白沉淀剂中,飞船上形成的所有晶体都为单晶,而地面上在多数沉淀剂中部生成大量孪晶.在相同沉淀剂中用液/液扩散法,飞船上生成CrFe蛋白的最大晶体比地面生成的最大晶体大1倍.而在相同沉淀剂中用汽相扩散的坐滴法,飞船上生成的MnFe蛋白最大晶体却没有地面生成的最大晶体大.这种差异也许是由不同结晶方法而不是不同蛋白所引起的.