Formation of a tight 1:1 complex of Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein: evidence for long-range interactions between the Fe protein binding sites during catalytic hydrogen evolution

It has been well documented that the combination of the MoFe protein of Azotobacter vinelandii nitrogenase (Av1) with the Fe protein (Cp2) from Clostridium pasteurianum nitrogenase produces an inactive, stable complex. However, we report that this heterologous nitrogenase has a low level of activity for H(2) evolution, with a specific activity of 12 nmol min(-)(1) mg(-)(1) of Av1. This activity does not arise from contaminating hydrogenase since it required the presence of both Cp2 and Av1 and showed saturation kinetics when increasing amounts of Cp2 were added to the assay. Incubation of the two proteins at a 4:1 Cp2:Av1 ratio in the absence of MgATP followed by analytical gel filtration showed, surprisingly, that the stoichiometry of the isolated complex was Av1.Cp2 instead of Av1.(Cp2)(2) as determined previously. The presence of MgATP in the elution buffer did not change the elution profile of the complex. The hydrodynamic radius of the isolated complex determined by dynamic light scattering was 5.93 +/- 0.14 nm, intermediate between Av1 and a stable 2:1 nitrogenase complex, consistent with a 1:1 assignment for the Av1.Cp2 complex. When assayed with Av2, the isolated Av1.Cp2 complex showed full half-site reactivity with a specific activity of 750 nmol of C(2)H(2) reduced min(-)(1) mg(-)(1) of Av1. The EPR spectrum of the isolated complex showed the Cp2 to be oxidized and the Av1 to retain the S = (3)/(2) signal characteristic of FeMoco. In the presence of MgATP, under turnover conditions at a 2:1 ratio of Cp2:Av1, the [4Fe-4S] center of Cp2 was protected from the chelator 2,2'-bipyridyl. This is consistent with the formation of a tight 2:1 complex of Av1.(Cp2)(2) which is more stable than the homologous Cp nitrogenase. Assuming that the Lowe-Thorneley model for nitrogenase applies and that a rate-limiting dissociation of the complex is required for H(2) evolution, then with a rate of 0.032 s(-)(1) the 1:1 complex is too stable to be involved in catalysis. The differences in the stability of the 2:1 and 1:1 complexes indicate cooperativity between the Fe protein binding sites of Av1, which structural data show to be separated by 105 A. On the basis of these observations, we propose a model for nitrogenase catalysis in which the stable 1:1 complex formed between oxidized Fe protein and the one-electron-reduced MoFe protein plays an essential role. In this scheme, the two Fe protein binding sites of the MoFe protein alternately bind and release Fe protein in a shuttle mechanism associated with long-range conformational changes in the MoFe 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.     

Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations.

Azotobacter vinelandii was grown diazotrophically at different dissolved oxygen concentrations (in the range of 3 to 216 microM) in sucrose-limited continuous culture. The specific nitrogenase activity, measured on the basis of acetylene reduction in situ, was dependent solely on the growth rate and was largely independent of oxygen and sucrose concentration. FeMo (Av1) and Fe (Av2) nitrogenase proteins were quantified after Western blotting (immunoblotting). When the cultures were grown at a constant dilution rate (D, representing the growth rate, mu) of 0.15.h-1, the cellular levels of both proteins were constant regardless of different dissolved oxygen concentrations. The same was true when the organisms were grown at D values above 0.15.h-1. At a lower growth rate (D = 0.09.h-1), however, and at lower oxygen concentrations cellular levels of both nitrogenase proteins were decreased. This means that catalytic activities of nitrogenase proteins were highest at low oxygen concentrations, but at higher oxygen concentrations they increased with growth rate. Under all conditions tested, however, the Av1:Av2 molar ratio was 1:(1.45 +/- 0.12). Cellular levels of flavodoxin and FeS protein II were largely constant as well. In order to estimate turnover of nitrogenase proteins in the absence of protein synthesis, chloramphenicol was added to cultures adapted to 3 and 216 microM oxygen, respectively. After 2 h of incubation, no significant decrease in the cellular levels of Av1 and Av2 could be observed. This suggests that oxygen has no significant effect on the breakdown of nitrogenase proteins.     

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.     

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.     

Nitrite, a new substrate for nitrogenase

We have examined the reactivity of the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2) toward nitrate and nitrite. Nitrate has no effect on H2 evolution or C2H2 reduction by nitrogenase and thus is neither a substrate nor an inhibitor. Nitrite dramatically inhibits H2 evolution. This inhibition has two components, one irreversible and one reversible upon addition of CO. The irreversible inhibition is due to nitrite inactivation of the Fe protein. The rate of this inactivation is greatly enhanced by addition of MgATP, suggesting the [4Fe-4S] cluster is the site of nitrite attack. The reversible inhibition does not represent an inhibition of electron flow but rather a diversion of electrons away from H2 evolution and into the six-electron reduction of nitrite to ammonia. Thus, nitrogenase functions as a nitrite reductase.     

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.     

Cross-linking of nitrogenase components. Structure and activity of the covalent complex

The nitrogenase complex from Azotobacter vinelandii is composed of the MoFe protein (Av1), an alpha 2 beta 2 tetramer, and the Fe protein (Av2), a gamma 2 dimer. During turnover of the enzyme, electrons are transferred from Av2 to Av1 in parallel with the hydrolysis of MgATP. Using the cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, we have identified some of the properties of the complex between the two components. The cross-linking reaction was highly specific yielding a single apparent Mr = 97,000 protein. The amount of cross-linked product was essentially independent of whether MgATP or MgADP were in the reaction. Also, the amount was maximum at high ratios of Av2 to Av1. The Mr = 97,000 protein was characterized by amino acid analysis and Edman degradation and was found to be consistent with a 1:1 complex of an Av2 gamma subunit and an Av1 beta subunit (the amino terminal serine subunit). The complex was no longer active in the nitrogenase reaction which supports, but does not prove, the requirement for dissociation of the complex after each electron transferred. Nitrogenase activity and cross-linking were inhibited in an identical way by NaCl, which suggests that electrostatic forces are critical to the formation of the electron transfer complex.     

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.     

Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis

Steady state kinetic measurements are reported for nitrogenase from Azotobacter vinelandii (Av) and Clostridium pasteurianum (Cp) under a variety of conditions, using dithionite as reductant. The specific activities of Av1 and Cp1 are determined as functions of Av2:Av1 and Cp2:Cp1, respectively, at component protein ratios from 0.4 to 50, and conform to a simple hyperbolic rate law for the interaction of Av2 with Av1 and Cp2 with Cp1. The specific activities of Av2 and Cp2 are also measured as a function of increasing Av1 and Cp1 concentrations, producing 'MoFe inhibition' at large MoFe:Fe ratios. When the rate of product formation under MoFe inhibited conditions is re-plotted as increasing Av2:Av1 or Cp2:Cp1 ratios, sigmoidal kinetic behavior is observed, suggesting that the rate constants in the Thorneley and Lowe (T&L) model are more dependent upon the oxidation level of MoFe protein than previously suspected [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 224 (1984) 887-894], at least when applied to Av and Cp. Calculation of Hill coefficients gave values of 1.7-1.9, suggesting a highly cooperative Fe-MoFe protein interaction in both Av and Cp nitrogenase catalysis. The T&L model lacks analytical solutions [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 215 (1983) 393-404], so the ease of its application to experimental data is limited. To facilitate the study of steady state kinetic data for H(2) evolution, analytical equations are derived from a different mechanism for nitrogenase activity, similar to that of Bergersen and Turner [Biochem. J. 131 (1973) 61-75]. This alternative cooperative model assumes that two Fe proteins interact with one MoFe protein active site. The derived rate laws for this mechanism were fitted to the observed sigmoidal behavior for low Fe:MoFe ratios (<0.4), as well as to the commonly observed hyperbolic behavior for high values of Fe:MoFe for both Av and Cp.     

Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 20.3) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Av1) were more complex than for Av2. A time-dependent inactivation of Av1 activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)     

M?ssbauer, EPR, and magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]1+ cluster with spin S = 3/2

We have studied the Fe protein (Av2) of the Azotobacter vinelandii nitrogenase system with M?ssbauer and EPR spectroscopies and magnetic susceptometry. In the oxidized state the protein exhibits M?ssbauer spectra typical of diamagnetic [4Fe-4S]2+ clusters. Addition of Mg.ATP or Mg.ADP causes a pronounced decline in the quadrupole splitting of the M?ssbauer spectra of the oxidized protein. Our studies show that reduced Av2 in the native state is heterogeneous. Approximately half of the molecules contain a [4Fe-4S]1+ cluster with electronic spin S = 1/2 and half contain a [4Fe-4S]1+ cluster with spin S = 3/2. The former yields the characteristic g = 1.94 EPR signal whereas the latter exhibits signals around g = 5. The magnetization of reduced Av2 is dominated by the spin S = 3/2 form of its [4Fe-4S]1+ clusters. These results explain a long standing puzzle, namely why the integrated spin intensity of the g = 1.94 EPR signal is substantially less than 1 spin/4 Fe atoms. In 50% ethylene glycol, 90% of the clusters are in the spin S = 1/2 form whereas, in 0.4 M urea, 85% are in the S = 3/2 form. In 0.4 M urea, the EPR spectrum of reduced Av2 exhibits well defined resonances at g = 5.8 and 5.15, which we assign to the S = 3/2 system. The EPR and M?ssbauer studies yield a zero-field splitting of 2D approximately equal to -5 cm-1 for this S = 3/2 state.     

Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases

Both heterologous crosses of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenase components are completely inactive, although the reasons for this incompatibility are not known. We have compared a number of properties of the MoFe proteins from these organisms (Cp1 and Av1, respectively) in an attempt to find differences that could explain this lack of functional activity. Optical and CD spectroscopic titrations are similar for both Av1 and Cp1, but EPR titrations are significantly different, suggesting different chemical reactivity patterns and/or magnetic interaction behavior. Similarly, reduction measurements on the six-electron-oxidized state of Cp1 and Av1 at controlled potentials indicate a difference in both the relative reduction sequence of the redox centers and the numerical values for their measured midpoint potentials. EPR measurements as a function of temperature also demonstrate that the relaxation behavior of the S = 3/2 MoFe centers associated with the proteins differ markedly. The Cp1 EPR signal only begins to undergo broadening above 65 K, whereas the Av1 signal is severely broadened above 25 K. These variations in the EPR properties for the two proteins are not likely to be due to differences in the stoichiometry and/or geometry of the MoFe cluster units themselves since similar EPR studies of the isolated cofactors showed them to be essentially identical. Thus, the different EPR behavior of the two proteins seems to arise either from protein constraints imposed on identical cofactors, and/or from magnetic interactions due to neighboring metal clusters.     

Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii

Wild-type Azotobacter vinelandii strain UW was transformed with plasmid pDB12 to produce a species (LS10) unable to synthesize the structural proteins of component 1 and component 2 of native nitrogenase. A spontaneous mutant of this strain was isolated (LS15) which can grow by nitrogen fixation in the presence or absence of either Mo or W. It is proposed that LS15 fixes nitrogen solely by an alternative nitrogen-fixing system which previously has been hypothesized to exist in A. vinelandii. Under nitrogen-fixing conditions, LS15 synthesizes a protein similar to component 2 (Av2) of native nitrogenase in that it can complement native component 1 (Av1) for enzymatic activity. Isolation and characterization of this second component 2 shows it to be a 4Fe-4S protein of molecular mass about 62 kDa and is antigenically similar to Av2. This protein is also similar to Av2 in that in the reduced state it possesses a rhombic ESR spectrum in the g = 2 region, which changes to an axial spectrum upon addition of MgATP. It is suggested that this second Fe-protein is associated with the alternative nitrogen-fixing system in A. vinelandii.     

棕色固氮菌缺失nifE的突变种固氮酶钼铁蛋白的结晶

从限氨固氮培养基中培养棕色固氮菌(Azotobacter vinelandii Lipmann)缺失nifE的突变种DJ35中,分离纯化得到缺失FeMoco的钼铁蛋白(ΔnifE Av1).在一定条件下结晶得到深棕色短斜四棱柱晶体.结晶溶液中各组分的浓度以及结晶方法等对其晶核数目、晶体大小和质量有明显影响.目前用气相扩散的悬滴法所得的最大晶体的二维边长分别为0.12 mm和0.13 mm.     

Hydrolysis of nucleoside triphosphates other than ATP by nitrogenase

The hydrolysis of ATP to ADP and P(i) is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron (Fe) protein and the molybdenum-iron (MoFe) protein. The oxidation state of nitrogenase was found to greatly influence the nucleotide hydrolysis rates. MgATP hydrolysis rates were 20 times higher under dithionite reducing conditions (approximately 4,000 nmol of MgADP formed per min/mg of Fe protein) as compared with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed per min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis rates were significantly higher under oxidizing conditions (1,400-2,000 nmol of MgNDP formed per min/mg of Fe protein) as compared with reducing conditions (80-230 nmol of MgNDP formed per min/mg of Fe protein). The K(m) values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330-540 microM) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF(4)(-) resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF(4)(-) x MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.     

The catalytic activity of nitrogenase in intact Azotobacter vinelandii cells

The influence of the growth conditions on the concentration of nitrogenase and on the nitrogenase activity, was studied in intact Azotobacter vinelandii cells. It was observed that whole cell nitrogenase activity could be enhanced in two ways. An increase of the growth rate of cells was accompanied by an increase in whole cell nitrogenase activity and by an increase in the concentration of nitrogenase in the cells. The molar ratio of Fe protein:MoFe protein was 1.47 +/- 0.17 and independent of the growth rate. Activity measurements in cell extracts showed that the catalytic activity of the nitrogenase proteins was independent of the growth rate of cells. The second way to increase whole cell nitrogenase activity was to expose cells to excess oxygen. Whole cells were exposed for 2.5 h to an enhanced oxygen-input rate. After this incubation nitrogenase activity was increased without an increase in protein concentration. It is calculated that the catalytic activity of the Fe protein in these cells was 6200 nmol C2H4 formed X min-1 X (mg Fe protein)-1. With these cells and with cells grown at a high growth rate, 50% of the whole cell activity is lost by preparing a cell-free extract. It will be demonstrated that this inactivation is partly caused by the activity measurements in vitro. When dithionite was replaced by flavodoxin as electron donor, a maximal catalytic activity of 4500 nmol C2H4 formed X min-1 X (mg Fe protein)-1 was measured in vitro for the Fe protein. The results are discussed in relation to the present model for nitrogenase catalysis.     

Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii

A new nitrogenase from Azotobacter vinelandii has been isolated and characterized. It consists of two proteins, one of which is almost identical with the Fe protein (component 2) of the conventional enzyme. The second protein (Av1'), however, has now been isolated and shown to differ completely from conventional component 1, i.e., the MoFe protein. This new protein consists of two polypeptides with a total molecular weight of around 200,000. In place of Mo and Fe it contains V and Fe with a V:Fe ratio of 1:13 +/- 3. The ESR spectrum of Av1' also differs from conventional component 1 in that lacks the g = 3.6 resonance that arises from the FeMo cofactor but contains an axial signal with gav less than 2 as well as inflections in the g = 4-6 region possibly arising from an S = 3/2 state. This new enzyme can reduce dinitrogen, protons, and acetylene but is only able to utilize 10-15% of its electrons for the reduction of acetylene.     

Properties of the MgATP and MgADP binding sites on the Fe protein of nitrogenase from Azotobacter vinelandii

Flow dialysis was used to study the binding of MgATP and MgADP to the nitrogenase proteins of Azotobacter vinelandii. Both reduced and oxidized Av2 bind two molecules of MgADP, with the following dissociation constants: reduced Av2, K1 = 0.091 +/- 0.021 mM and K2 = 0.044 +/- 0.009 mM; oxidized Av2, K1 = 0.024 +/- 0.015 mM and K2 = 0.039 +/- 0.022 mM. Binding of MgADP to reduced Av2 shows positive co-operativity. Oxidized Av2 binds two molecules of MgATP with dissociation constants K1 = 0.049 +/- 0.016 mM and K2 = 0.18 +/- 0.05 mM. Binding data of MgATP to reduced Av2 can be fitted by assuming one binding site, but a better fit was obtained by assuming two binding sites on the protein with negative co-operativity and with dissociation constants K1 = 0.22 +/- 0.03 mM and K2 = 1.71 +/- 0.50 mM. It was found that results concerning the number of binding sites and the dissociation constants of MgATP-Av2 and MgADP-Av2 complexes depend to a great extent on the specific activity of the Av2 preparation used, and that it is difficult to correct binding data for inactive protein. No binding of MgADP to Av1 could be demonstrated. Binding studies of MgADP to a mixture of Av1 and Av2 showed that Av1 did not affect the binding of MgADP to either oxidized or reduced Av2. Inhibition studies were performed to investigate the interaction of MgATP and MgADP binding to oxidized and reduced Av2. All the experimental data can be explained by the minimum hypothesis, i.e. the presence of two adenine nucleotide binding sites on Av2. MgATP and MgADP compete for these two binding sites on the Fe protein.     

Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene codes for 130-kDa toxin protein

Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene from Bacillus thuringiensis var. kurstaki borne on pKT230, shuttle vector, was generated. PCR amplification of Cry1Ac gene present in recombinant G. diazotrophicus yielded a 278-bp DNA product. The nitrogenase assay has revealed that the recombinant G. diazotrophicus in sugarcane stem produced similar levels of nitrogenase compared to wild-type G. diazotrophicus. The presence of 130-kDa protein in apoplastic fluid from sugarcane stem harvested from pots inoculated with recombinant G. diazotrophicus shows that the translocated G. diazotrophicus produces 130-kDa protein which is recognized by the hyperimmune antiserum raised against 130-kDa protein. The first instar Eldana saccharina neonate larvae that fed on artificial medium containing recombinant G. diazotrophicus died within 72 h after incubation.