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 vinela...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.展开更多
Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from Δ nifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann....Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from Δ nifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann. Systematic studies on the effect of concentrations of PEG 8000,MgCl 2, NaCl,Tris and buffer pH on the crystallization and crystal growth of the protein showed that the protein could not be crystallized in lower concentrations of the chemicals and lower buffer pH. A large amount of smaller crystals of the protein appeared in a week with gradual increasing in the chemical concentrations and pH≥8.0. When the chemical concentrations were further increased, the time for crystallization was increased and a few high grade crystals of larger size were formed. If the concentrations of the chemicals were continuously increased, many crystals with smaller size, and, sometimes of poor quality appeared again and eventually ceased to produce any crystals. The optimal concentration for each of the above mentioned chemicals varies with other variable factors. Only one bigger crystal (both of the longest two sides: 0.16 mm) could be obtained in a hanging drop of protein sample when the concentrations of PEG 8000, MgCl 2, NaCl,Tris and protein were kept at 1.86%, 300 mmol/L, 400 mmol/L, 53 mmol/L and 4.64 g/L , respectively, with Tris buffer pH 8.2.展开更多
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 Lipman...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.展开更多
It is reported that fermentative liquids with various concentrations of La and Nd affect the fer- mentation of alginic acid from the strain 342 of Azotobacter vinelandii.The results are as follows:When the concentrati...It is reported that fermentative liquids with various concentrations of La and Nd affect the fer- mentation of alginic acid from the strain 342 of Azotobacter vinelandii.The results are as follows:When the concentration of La or Nd was up to 100 ppm,the cell growth is stimulated and the production of alginic acid is promoted.The La or Nd in concentration higher than 200 ppm or 150 ppm inhibits the fermentation, respectively.As the concentration range of La is 0～100 ppm or that of Nd is 0～150 ppm,the yield of fixed nitrogen increases,and the ratio of c_M to c_G(c_M/c_G)decreases with the raise of the concentration of La or Nd.When the concentration range of La is 100～400 ppm and that of Nd is 150～400 ppm,the conclusion is contrary to the above mentioned result.展开更多
Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (?nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). ...Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (?nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). The results of Western blotting after anoxic native electro-phoresis and SDS-PAGE showed that ?nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Fur-thermore, ?nifZ Av1 was also similar to OP Av1 at the mo-lybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (?ε) of circular dichroism (CD) at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the ?nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and sub-strate (C2H2, H+ and N2)-reduction activity of ?nifZ Av1 were 74% and 46%―50% of those of OP Av1, respectively. Fur-thermore, the ?ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between ?nifZ Av1 and OP Av1 resulted from P-cluster rather than FeMoco, and from the half num-ber of P-cluster in ?nifZ Av1, but the composition or redox state of P-cluster in ?nifZ Av1 were not changed. Thus it could propose that ?nifZ Av1 is composed of two different αβ subunit pairs. One is a FeMoco- and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-con- taining pair. Since the deletion of nifZ gene leads to the defi-ciency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.展开更多
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 de...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.展开更多
A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization tim...A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization time-of-flight mass spectrometry, the protein was Identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra （ABS） In the reduced state exhibited three peaks at 421,517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichrolsm （CD） spectrum of HBP59 In the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the △ε （CD extinction coefficient） values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, -0.65 and -1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully Interacted with heme, Its maximal ABS value and Soret CD Intensity were increased by approximately 10-fold compared with values before Interaction. Therefore, It seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be Involved In heme utilization or adhesion.展开更多
A MoFe protein (ΔnifE Av1) with a purity of ～80% was purified from a nifE-deleted mutant of Azotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP (OP Av1), ΔnifE Av1 had the same...A MoFe protein (ΔnifE Av1) with a purity of ～80% was purified from a nifE-deleted mutant of Azotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP (OP Av1), ΔnifE Av1 had the same subunits composition, and had immune reaction with antibody to OP Av1, but its relative mobility in anaerobic native polyacrylamide gel electrophoresis (PAGE) was a little larger than that of OP Av1. Metal analysis showed that Mo and Fe contents of ΔnifE Av1 both apparently decreased. When complemented with OP Fe protein, ΔnifE Av1 had no C2H2-reduction activity, but it could be in vitro activated by FeMoco extracted from OP Av1. The circular dichroism (CD) spectrum of ΔnifE Av1 at ~450 nm was similar to that of OP Av1, while the EPR signal at g≈3.7 was absolutely silent, and the signal intensities at g≈4.3 and 2.0 decreased by 75% and 50%, respectively. The results indicated that ΔnifE Av1 purified from DJ35 was a FeMoco-deficient but P-cluster-con- taining MoFe protein.展开更多
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...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.展开更多
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 growt...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.展开更多
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 gro...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.展开更多
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...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.展开更多
文摘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.
文摘Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from Δ nifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann. Systematic studies on the effect of concentrations of PEG 8000,MgCl 2, NaCl,Tris and buffer pH on the crystallization and crystal growth of the protein showed that the protein could not be crystallized in lower concentrations of the chemicals and lower buffer pH. A large amount of smaller crystals of the protein appeared in a week with gradual increasing in the chemical concentrations and pH≥8.0. When the chemical concentrations were further increased, the time for crystallization was increased and a few high grade crystals of larger size were formed. If the concentrations of the chemicals were continuously increased, many crystals with smaller size, and, sometimes of poor quality appeared again and eventually ceased to produce any crystals. The optimal concentration for each of the above mentioned chemicals varies with other variable factors. Only one bigger crystal (both of the longest two sides: 0.16 mm) could be obtained in a hanging drop of protein sample when the concentrations of PEG 8000, MgCl 2, NaCl,Tris and protein were kept at 1.86%, 300 mmol/L, 400 mmol/L, 53 mmol/L and 4.64 g/L , respectively, with Tris buffer pH 8.2.
文摘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.
文摘It is reported that fermentative liquids with various concentrations of La and Nd affect the fer- mentation of alginic acid from the strain 342 of Azotobacter vinelandii.The results are as follows:When the concentration of La or Nd was up to 100 ppm,the cell growth is stimulated and the production of alginic acid is promoted.The La or Nd in concentration higher than 200 ppm or 150 ppm inhibits the fermentation, respectively.As the concentration range of La is 0～100 ppm or that of Nd is 0～150 ppm,the yield of fixed nitrogen increases,and the ratio of c_M to c_G(c_M/c_G)decreases with the raise of the concentration of La or Nd.When the concentration range of La is 100～400 ppm and that of Nd is 150～400 ppm,the conclusion is contrary to the above mentioned result.
基金supported by the State Key Basic Research and Developmental Plan of China(Grant No.001CB1089-06)the National Natural Science Foundation of China(Grant No.30270296)the National Science Foundation for distinguished Young Scholars of China(Grant No.20403024).
文摘Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (?nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194). The results of Western blotting after anoxic native electro-phoresis and SDS-PAGE showed that ?nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Fur-thermore, ?nifZ Av1 was also similar to OP Av1 at the mo-lybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (?ε) of circular dichroism (CD) at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the ?nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and sub-strate (C2H2, H+ and N2)-reduction activity of ?nifZ Av1 were 74% and 46%―50% of those of OP Av1, respectively. Fur-thermore, the ?ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between ?nifZ Av1 and OP Av1 resulted from P-cluster rather than FeMoco, and from the half num-ber of P-cluster in ?nifZ Av1, but the composition or redox state of P-cluster in ?nifZ Av1 were not changed. Thus it could propose that ?nifZ Av1 is composed of two different αβ subunit pairs. One is a FeMoco- and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-con- taining pair. Since the deletion of nifZ gene leads to the defi-ciency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.
基金Supported by the State Key Basic Research and Development Plan of China （001CB1089-06） and the National Natural Science Foundation of China （30270296）.
文摘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.
基金Supported by the State Key Basic Research and Development Plan of China （001CB1089-06） and the National Natural Science Foundation of China （30270296）.Acknowledgements The authors thank Professors YX Jing and JG Li for their valuable advice.
文摘A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization time-of-flight mass spectrometry, the protein was Identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra （ABS） In the reduced state exhibited three peaks at 421,517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichrolsm （CD） spectrum of HBP59 In the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the △ε （CD extinction coefficient） values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, -0.65 and -1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully Interacted with heme, Its maximal ABS value and Soret CD Intensity were increased by approximately 10-fold compared with values before Interaction. Therefore, It seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be Involved In heme utilization or adhesion.
基金supported by the State Key Basic Research and Developmental Plan of China(Grant No.001CB1089-06)the National Natural Science Foundation of China(Grant No.30270296)the National Science Foundation for Distin-guished Young Scholars of China(Grant No.20403024).
文摘A MoFe protein (ΔnifE Av1) with a purity of ～80% was purified from a nifE-deleted mutant of Azotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP (OP Av1), ΔnifE Av1 had the same subunits composition, and had immune reaction with antibody to OP Av1, but its relative mobility in anaerobic native polyacrylamide gel electrophoresis (PAGE) was a little larger than that of OP Av1. Metal analysis showed that Mo and Fe contents of ΔnifE Av1 both apparently decreased. When complemented with OP Fe protein, ΔnifE Av1 had no C2H2-reduction activity, but it could be in vitro activated by FeMoco extracted from OP Av1. The circular dichroism (CD) spectrum of ΔnifE Av1 at ~450 nm was similar to that of OP Av1, while the EPR signal at g≈3.7 was absolutely silent, and the signal intensities at g≈4.3 and 2.0 decreased by 75% and 50%, respectively. The results indicated that ΔnifE Av1 purified from DJ35 was a FeMoco-deficient but P-cluster-con- taining MoFe protein.
基金supported by the State Key Basic Research and Development Plan of China(Grant No.001CB1089-06)the National Science Foundation for Distinguished Young Scholars of China(Grant No.20403024)the National Natural Science Foundation of China(Grant No.30270296).
文摘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.
文摘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.
文摘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.
文摘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.
