Crystallization of Nitrogenase MoFe Protein from a Mutant nifE Deleted Strain of Azotobacter vinelandii

Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from FeMoco-deficient MoFe protein (DeltanifE Avl) purified from a nifE deleted mutant DJ35 of Azotobacter vinelandii Lipmann grown in NH3-limited medium. The number, size and quality of crystals were significantly affected by either the concentration of precipitants and buffer or diffusion method. The longest sides of the largest crystal of DeltanifE Avl protein, which was obtained by vapor diffusion in the hanging drop method, were 0.12 and 0.13 mm, respectively.

Growth of the Crystals of Nitrogenase MnFe Protein

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 Delta nifZ 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 Characteristics of Mn-containing Nitrogenase Component Ⅰ

A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but undergo phenotypic reversal to Nif + under Mo deficiency, was able to grow in Mo- and NH 3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW 3 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 C 2H 2- 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 Ⅰ protein.

Purification and Characterization of Cr-containing Nitrogenase Component Ⅰ

A mutant UW 3, which is unable to fix N 2 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 NH 3_deficient medium. A partly purified nitrogenase component Ⅰ protein obtained from UW 3 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 C 2H 2_ 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 different subunits (α 2β 2). The preliminary results indicated that the Cr_containing protein might be a nitrogenase component Ⅰ protein.

Crystal Growth of Nitrogenase CrFe Protein and MnFe Protein in Space

Nitrogenase CrFe protein and MnFe protein were purified from a mutant strain UW3 of Azotobacter vinelandii Lipmann grown on a medium containing Cr and Mn, respectively. In order to meet the requirement for crystal growth Of O-2-susceptible proteins including nitrogenase in space, crystallization conditions were optimized for the proteins using a simple and suitable device, as a replacement for the cumbersome anaerobic box (dry box), for anaerobic addition of the protein samples. In all used precipitant and protein solutions added in the simplified plexi glass box, CrFe protein and MnFe protein could be crystallized on the spacecraft in one week by the liquid/liquid diffusion method and vapor diffusion by the sitting drop method, respectively. All formed crystals were single on the spacecraft, but under the same condition twin crystals appeared on the ground. The size of the largest crystal grown in space from CrFe protein was 2-fold larger than that on the ground. But the size of the largest crystal grown in space from MnFe protein was not larger than that on the ground. The difference in crystal growth in space between CrFe protein and MnFe protein could be resulted from the crystallization method, rather than the kind of protein.

Growth of Large Single Crystals of Nitrogenase CrFe Protein and MnFe Protein

By using the liquid/liquid diffusion method at a suitable crystallization conditions, large single and dark brown crystals (the sides of the largest crystals were 0.20 mm x 0.20 mm x 0.07 min and 0.18 mm x 0.18 mm x 0.05 mm, respectively) could be obtained from the solutions of nitrogenase CrFe protein and MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmarm grown in Cr- or Mn-containing but NH3-free medium. The time of crystal formation, as well as the number, size, shape and quality of crystals obviously depended on the concentrations of PEG, MgCl2 and NaCl. The liquid/liquid diffusion method seems to benefit CrFe protein and MnFe protein for the growth of large single crystals for X-ray diffraction analysis.

Crystalline Growth of Nitrogenase CrFe Protein

Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Cr-containing but NH3-free Medium, The possibility of crystallization, as well as the. number, size and quality of crystals obviously depended on the concentrations of PEG 8000, MgCl2, NaCl, Tris and Hepes buffer, and methods of crystallization. The optimum concentrations of the chemicals for crystallization of CrFe protein were slightly different from those for crystallization of MnFe protein from UW3 grown in Mn and DeltanifZ MoFe protein from a nifZ deleted strain of A. vinelandii. The crystal seemed to be formed from CrFe protein.

Crystallization of Nitrogenase MoFe Protein (NifB Av1) from a nifB Mutated Strain UW45 of Azotobacter vinelandii

Six hundred and 28 mg of NifB(-) Av1 was obtained by a chromatography twice on DE 52 columns and Sephacryl S-300 column from the crude extract (37 677 mg) of a nifB mutated strain UW45 of Azotobacter vinelandii Lipmann. The protein was almost homogeneous as determined by Coomassie staining of SDS gels. The analysis by SDS-PAGE showed that NifB(-)Av1 was similar to Av1 from wild-type strain of A. vinelandii (OP) in the kinds of subunits (alpha and beta subunit). When complemented with Av2, NifB(-)Av1 had hardly any H-reducing activity, but could be significantly activated by FeMoco extracted from Av1. Under a suitable condition for crystallization, short dark-brown rhombohedral crystals could be obtained from NifB(-)Av1. Both of the longest sides of the biggest crystal were 0.1 mm. The time of the formation of crystals and number, size, quality and shape of crystals obviously depended not only on the kinds and concentrations of the components in the precipitant solution, but also on the methods for crystallization and technical bias, etc. The preliminary results showed that the crystal seemed to be formed from NifB(-)Av1.

Studies on Chem icalCom position ofIron-m olybdenum Cofactor fromNitrogenase by Fluorescence Analysis

The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence intensity of fluorescein dimercury acetate(FDMA)(in 1 mol/L NaOH) were investigated by fluorophotometric titrations and compared with those of (NH 4) 2MoS 4 and complexes of (NH 4) 2MoS 4 with Na 2S or Na 2S 2 or (NH 4) 2S x on the relative properties of FDMA. It was found that the electronic spectrum of FDMA displayed hypsochromic shift(17 nm) in the presence of FeMoco just like that in the presence of other inorganic sulfides and that the titration curve for the quench of FDMA with FeMoco is very similar to that for the quench of FDMA with complex of (NH 4) 2MoS 4 with Na 2S 2(mole ratio is 1∶3). The results showed that FeMoco(N) probably contained S—S bonds and its structure was found to be changed compared with Kim Rees structural model. This change should profit the formation of the polymer.

SYNTHESIS AND STRUCTURE OF A LINEAR TRIVANADIUM COMPOUND(Ph_4P)[V_3(OC_6H_4S-o)_6]-SIMULATION OF THE VANADIUM SITE OF ALTERNATIVE NITROGENASE

Compound(Ph_4P)[V_3MP_6](MPH_2=o-HOC_6H_4SH)was obtained by reaction of VCl_3 and Na_2MP in ethanol in the presence of Ph_4PBr.It is triclinic and crystallizes in space group P1,fw=1237.3,a=14.127(4), b=14.342(4),c=15.878(4);α=65.08(2),β=73.09(2),T=78.68(2)°;V=2781.3~3, Z=2,d_c=1.48 g/cm^3.Final R factor is 0.063.The three vanadium atoms are linearly arranged and bridged by the oxygen atoms and terminally chelated by the thiolato-atoms of the six MP^2-ligands in pseudo-S_6 symmetry.

Diversity of Nitrogenase Systems in Diazotrophs

Nitrogenase is a metalloprotein complex that catalyses the reaction of biological nitrogen fixation. At least three genetically distinct nitrogenase systems have been confirmed in diazotrophs, namely Nil, Vnf, and Anf, in which the active-site central metals are Mo, V, and Fe, respectively. The present review summarizes progress on the genetic, structural, and functional investigations into the three nitrogenases and discusses the possibility of the existence of other novel nitrogenases.

Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.

Retrospect and prospect of some proposed 'string-bag' structure models for the active center of nitrogenase

The problem of how the nitrogenase is capable of transforming dinitrogen into ammoniahas indeed become one of the hot subjects in the field of molecular biochemistry. In1992, Kim and Rees of the California Institute of Technology published their result of

Reconstitution activity of partial metallocluster-deficient molybdenum-iron protein of nitrogenase with a series of molybdenum-sulfur compounds

RECENTLY,the structure of MoFe protein from Azotobacter vinelandii has been deter-mined,and based on this,the structural models of FeMo-co and P-clusters in MoFe proteinhave been proposed.According to the model,FeMo-co consists of two clusters,Fe4S3 andMoFe3S3 with a homocitrate ligand coordinated to Mo atom,bridged by three nonprotein lig-ands,two of which are inorganic sulfur atoms and the other is nitrogen- or

The influence of desiccation on the recovery process of nitrogenase activity in restored biological soil crusts

Dear Editor,Biological soil crusts(BSCs),a layered structure formed by associations of soil organisms and topsoil,dominate arid and semiarid areas and serve important ecological functions in these areas(Eldridge and Greene,1994).Nitrogen fixation by BSCs is the main source of N in arid and semi-arid ecosystems.Desiccation is the most notable factor that influences BSCs,which recover physiological activity only after moistening.By influencing the amount of carbohydrates,

Structural comparison to the molybdenumsite of nitrogenase Synthesis and crystal structure of binuclear oxomolybdenum(V)complex with both omercaptophenolate and alkoxide ligands

Complex(n-Bu_4N)[Mo_2O_2(OMe)(mp)_3][1,mp=(o-OC_(?)H_4S)^(2-)]was synthesized by the reaction of MoOCl_3(THF)_2 and Na_2mp in EtOH and crystallized in monoclinic space group P2_(?)/n with crystal data:a=13.910(3),b=23.554(3),c=12.558(2)(?),β=105.20(2)°,Z=4,D_o=1.455g/ cm^3,final R=0.077 for 5325 reflections[I>3σ(I)].The two[Mo(O)mp]^+ moicties are bridged by one 1,2-bidentate ligand mp^(2-) with exceedingly small bite angle(69.0°)and bite distance (2.70(?))and by a methoxy group.The structure can be interpreted as two distorted octahedra around the Mo atoms sharing a face.Attempts have been made to provide structural informa- tions for the syntheses of modelling compounds of FeMoco by comparing the Mo—O and Mo—S bond distances of o-mercaptophenolate ligated compounds to those of the molybdenum site of nitrogenase.

Effect of microbial agent ARC-BBBE demonstration application on peanut production in the Huang-huai-hai area of China

The microbial agent ARC-BBBE demonstration trials were conducted in four provinces in the main peanutproducing areas of the Huang-huai-hai plain of China.The results revealed that the application of ARC-BBBE led to a 1.09–1.70 fold increase in the number of nodules in the treatment group at the demonstration site compared to the control group.Moreover,the nodule weight in the treatment group was 0.80–3.32 times higher than that of the control group,and nitrogenase activity per plant showed a significant enhancement by 1.00–2.83 fold compared to controls.Additionally,notable improvements were observed in terms of increased fresh weight of whole plants,well-filled pod numbers,and enhanced growth performance;ultimately resulting in a harvest yield increase ranging from 9.46%to 49.04%.The abundance of Aspergillus flavus in rhizosphere soil was determined by the dilution spread plate method,and the inhibition rate was up to 86.7%.The application of ARC-BBBE in the significant peanut-producing areas of Huang-huai-hai has effects of promoting growth,nodulation,and increasing production.At the same time,it has the effect of inhibiting and controlling soil Aspergillus flavus,which provides a new green and low-carbon way to promote the high-quality development of the peanut industry.

Effect of Water Stress and Foliar Boron Application on Seed Protein, Oil, Fatty Acids, and Nitrogen Metabolism in Soybean

Effects of water stress and foliar boron (FB) application on soybean (Glycine max (L) Merr.) seed composition and nitrogen metabolism have not been well investigated. Therefore, the objective of this study was to investigate the effects of water stress and FB on seed protein, oil, fatty acids, nitrate reductase activity (NRA), and nitrogenase activity (NA). A repeated greenhouse experiment was conducted where one set of soybean plants were subjected to water stress (WS), and the other set was watered (W). Foliar boron (B) was applied at rate of 0.45 kg·ha-1. Treatments were watered-plants with no FB (W), watered-plants with FB (WB), water-stress plants with no FB (WS), and water-stress plants with FB (WSB). The results showed that seed protein and oil percentage were significantly (P 15N/ 14N and 13C/12C natural abundance were altered between watered-and watered-stressed plants. These results suggest that water stress and FB can influence seed composition, and nitrogen metabolism, and 15N/14N and 13C/12C ratios, reflecting environmental and metabolic changes in carbon and nitrogen fixation pathways. Lack of B translocation from leaves to seed under water stress may suggest a possible mechanism of limited B translocation under water stress. These findings may be beneficial to breeders to select for B translocation efficiency under drought conditions. Altered 15N/14N and 13C/12C under water stress can be used as a tool to select for drought tolerance using N and C isotopes in the breeding programs.

Solid and Solution Structural Studies of Dimeric and Tetrameric Molybdenum(VI) Malate Complexes

Reactions of potassium molybdate with racemic malic acid （H3mal = C4H6O5） result in the isolation of two mesomeric molybdenum malate complexes K8[（MoO2）2O（R-mal）2][（MoO2）2O（Smal）2]-4H2O 1 and （Him）2K6[（MoO2）4O3（R-mal）2][（MoOE）4O3（S-mal）2]-8H2O 2. Complex 1 belongs to the monoclinic system, space group C2/c with a = 14.8637（3）, b = 6.9544（1）, c = 19.6783（5）A, β = 100.081（2）°, V = 2002.70（7） A^3, Mr = 1452.88, Z = 2, F（000） = 1416, T = 173 K, Dc = 2.409 g/cm3, fl（MoKa＇） = 2.167, R = 0.0283 and wR = 0.0733.2 is of triclinic system, space group P1^- with a = 8.7707（2）, b = 9.3310（3）, c = 17.9093（7）A, α= 83.781（3）, β = 85.626（2）, y= 84.822（2）°, V = 1447.84（8）A^3, Mr = 2160.68, Z = 1, F（000） = 1048, T = 173 K, Dc = 2.478 g/cm^3,μ（MoKα） = 2.230, R = 0.0234 and wR = 0.0584.1 is the first isolated dinuclear molybdenum（VI） malato complex in 1：1 molar ratio. The molybdenum atoms in the two complexes are six-coordinated in an approximately octahedral geometry. Two malates coordinate tridentately with the Mo atom via their α-alkoxy, α-carboxy and α-carboxy groups in 1 and 2. β-Carboxy group in 2 further links with the other two Mo atoms to give a tetrameric unit. The solution ^1H and ^13C NMR spectra indicate that dimeric malate molybdenum in 1 dissociates partly in solution and exists in an equilibrium with tetrameric species, while 2 is stable and retains its tetrameric structure without any dissociation.

“Biological Nitrogen Fixation” Book Summary

Biological nitrogen fixation is a very valuable alternative to nitrogen fertilizer. This process will be discussed in the “Biological Nitrogen Fixation” book. A wide array of free-living and associative nitrogen fixing organisms (diazotrophs) will be covered. The most extensively studied and applied example of biological nitrogen fixation is the symbiotic interaction between nitrogen fixing “rhizobia” and legume plants. While legumes are important as major food and feed crops, cereals such as wheat, maize and rice are the primary food crops, but do not have this symbiotic nitrogen fixing interaction with rhizobia. It has thus been a “holy grail” to transfer the ability to fix nitrogen to the cereals and this topic will be also addressed in these books.