采用 52℃下加热 6 min,后经 DEAE- 52、Sephacryls S- 2 0 0和 Q- Sepharose等柱层析方法 ,分离纯化了棕色固氮菌 (Azotobacter vinelandii)缺失 nif Z基因突变种固氮酶 Mo Fe(Δnif Z Mo Fe)蛋白 ,其纯度达到电泳纯。Δnif Z Mo Fe蛋...采用 52℃下加热 6 min,后经 DEAE- 52、Sephacryls S- 2 0 0和 Q- Sepharose等柱层析方法 ,分离纯化了棕色固氮菌 (Azotobacter vinelandii)缺失 nif Z基因突变种固氮酶 Mo Fe(Δnif Z Mo Fe)蛋白 ,其纯度达到电泳纯。Δnif Z Mo Fe蛋白的固氮活性为 2 83nmol C2 H2 还原 / (min·mg蛋白 ) ,远低于野生种 Mo Fe蛋白。Δnif Z Mo Fe蛋白对氧更敏感 ;热稳定性略低于野生种。Δnif Z Mo Fe蛋白的可见光吸收光谱与野生种 Mo Fe蛋白极为相似。其圆二色谱和磁圆二色谱在 450～ 550 nm与野生种 Mo Fe蛋白显著不同 ,表明其 P- cluster及其周围环境与野生种 Mo Fe蛋白有所差异。这亦可能是造成缺失 nif Z突变种 Mo Fe蛋白固氮活性低的原因。展开更多
Single cubane-like clusters, MFe<sub>3</sub>S<sub>4</sub>（R<sub>2</sub>dtc）<sub>5</sub> （M=Mo, R<sub>2</sub>=—（CH<sub>2</sub>）<sub>4&...Single cubane-like clusters, MFe<sub>3</sub>S<sub>4</sub>（R<sub>2</sub>dtc）<sub>5</sub> （M=Mo, R<sub>2</sub>=—（CH<sub>2</sub>）<sub>4</sub>—, Bz<sub>2</sub>; M=W, R<sub>2</sub>=Me<sub>2</sub>, —（CH<sub>2</sub>）4—; dtc=dialkyldithiocarbamate） were synthesized by assembly reaction of （NH<sub>4</sub>）<sub>2</sub>MS<sub>4</sub> （M=Mo, W）, FeCl<sub>2</sub> with R<sub>2</sub>dtcNa （or NH<sub>4</sub>） in DMF. The structure, IR, EPR and cyclic voltammogram are described and discussed. MoFe<sub>3</sub>S<sub>4</sub>（C<sub>4</sub>H<sub>8</sub>dtc）<sub>5</sub> was also obtained from the conversion of linear cluster Fe（DMF）<sub>6</sub>[（FeCl<sub>2</sub>）<sub>2</sub>MoS<sub>4</sub>] in the presence of C<sub>4</sub>H<sub>8</sub>dtc NH<sub>4</sub>. Both clusters MoFe<sub>3</sub>S<sub>4</sub>（C<sub>4</sub>H<sub>8</sub>dtc）<sub>4</sub>·2C<sub>2</sub>H<sub>5</sub>CN and WFe<sub>3</sub>S<sub>4</sub>（C<sub>4</sub>H<sub>8</sub>dtc）<sub>5</sub>·2DMF crystallize in the monoclinic space group C2/c with a=12.544（2）, （Mo-cluster）, 12.533（4） （W-cluster）; b=20.478（4）, 20.121（6）; c=19.582（7）, 19.659（2）;β=102.82（2）, 102.96（3）°; and Z=4, 4. Their structures were refined to R=0.057 and 0.064 with use of 2690 and 2179 reflections （ι】3Q（I））, respectively. Many similarities in structure for Mo and W clusters indicate that both Mo and W play a very similar role in the clusters.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
The features of synthetic processes are described on the basis of the studies of the syntheses of MoFe<sub>3</sub>S<sub>4</sub> cubane-like cluster compounds either by self-assembly reactions o...The features of synthetic processes are described on the basis of the studies of the syntheses of MoFe<sub>3</sub>S<sub>4</sub> cubane-like cluster compounds either by self-assembly reactions or by the conversion of linear clusters. The redox properties and the structure of ligands as well as the stoichiometry of reactants have obvious effects upon the core oxidation state and the structure of products. It is indicated that the self-assembly reaction may pass through a combination process oflinear cluster intermediates having structural unit （M,M’=Mo） Fe).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
In the study of the catalytic active center of nitrogen fixation in nitrogenase, structural models containing MoFe3S3 or MoFe3S4 cubane-like skeleton have been proposed. The syntheses of these model compounds have sin...In the study of the catalytic active center of nitrogen fixation in nitrogenase, structural models containing MoFe3S3 or MoFe3S4 cubane-like skeleton have been proposed. The syntheses of these model compounds have since attracted the attention of many chemists. Up to the present time, however, only Holm’s research group has obtained MoFe3S4 single cubane cluster compounds by multi-step cleavage reaction of Mo—Fe—S展开更多
老年多脏器功能衰竭（multipleorgan failure in the elderly,MOFE）发病机制复杂,病情凶险,进展迅速,治疗困难,病死率高。现对1998年1月至2008年10月我院480例MOFE患者进行回顾性分析，旨在探讨MOFE发生的相关因素，以期对临床预防和...老年多脏器功能衰竭（multipleorgan failure in the elderly,MOFE）发病机制复杂,病情凶险,进展迅速,治疗困难,病死率高。现对1998年1月至2008年10月我院480例MOFE患者进行回顾性分析，旨在探讨MOFE发生的相关因素，以期对临床预防和治疗提供参考，报告如下。展开更多
文摘采用 52℃下加热 6 min,后经 DEAE- 52、Sephacryls S- 2 0 0和 Q- Sepharose等柱层析方法 ,分离纯化了棕色固氮菌 (Azotobacter vinelandii)缺失 nif Z基因突变种固氮酶 Mo Fe(Δnif Z Mo Fe)蛋白 ,其纯度达到电泳纯。Δnif Z Mo Fe蛋白的固氮活性为 2 83nmol C2 H2 还原 / (min·mg蛋白 ) ,远低于野生种 Mo Fe蛋白。Δnif Z Mo Fe蛋白对氧更敏感 ;热稳定性略低于野生种。Δnif Z Mo Fe蛋白的可见光吸收光谱与野生种 Mo Fe蛋白极为相似。其圆二色谱和磁圆二色谱在 450～ 550 nm与野生种 Mo Fe蛋白显著不同 ,表明其 P- cluster及其周围环境与野生种 Mo Fe蛋白有所差异。这亦可能是造成缺失 nif Z突变种 Mo Fe蛋白固氮活性低的原因。
基金Project supported by the National Natural Science Foundation of China and the Science Foundation of Academia Sinica.
文摘Single cubane-like clusters, MFe<sub>3</sub>S<sub>4</sub>（R<sub>2</sub>dtc）<sub>5</sub> （M=Mo, R<sub>2</sub>=—（CH<sub>2</sub>）<sub>4</sub>—, Bz<sub>2</sub>; M=W, R<sub>2</sub>=Me<sub>2</sub>, —（CH<sub>2</sub>）4—; dtc=dialkyldithiocarbamate） were synthesized by assembly reaction of （NH<sub>4</sub>）<sub>2</sub>MS<sub>4</sub> （M=Mo, W）, FeCl<sub>2</sub> with R<sub>2</sub>dtcNa （or NH<sub>4</sub>） in DMF. The structure, IR, EPR and cyclic voltammogram are described and discussed. MoFe<sub>3</sub>S<sub>4</sub>（C<sub>4</sub>H<sub>8</sub>dtc）<sub>5</sub> was also obtained from the conversion of linear cluster Fe（DMF）<sub>6</sub>[（FeCl<sub>2</sub>）<sub>2</sub>MoS<sub>4</sub>] in the presence of C<sub>4</sub>H<sub>8</sub>dtc NH<sub>4</sub>. Both clusters MoFe<sub>3</sub>S<sub>4</sub>（C<sub>4</sub>H<sub>8</sub>dtc）<sub>4</sub>·2C<sub>2</sub>H<sub>5</sub>CN and WFe<sub>3</sub>S<sub>4</sub>（C<sub>4</sub>H<sub>8</sub>dtc）<sub>5</sub>·2DMF crystallize in the monoclinic space group C2/c with a=12.544（2）, （Mo-cluster）, 12.533（4） （W-cluster）; b=20.478（4）, 20.121（6）; c=19.582（7）, 19.659（2）;β=102.82（2）, 102.96（3）°; and Z=4, 4. Their structures were refined to R=0.057 and 0.064 with use of 2690 and 2179 reflections （ι】3Q（I））, respectively. Many similarities in structure for Mo and W clusters indicate that both Mo and W play a very similar role in the clusters.
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.
文摘The features of synthetic processes are described on the basis of the studies of the syntheses of MoFe<sub>3</sub>S<sub>4</sub> cubane-like cluster compounds either by self-assembly reactions or by the conversion of linear clusters. The redox properties and the structure of ligands as well as the stoichiometry of reactants have obvious effects upon the core oxidation state and the structure of products. It is indicated that the self-assembly reaction may pass through a combination process oflinear cluster intermediates having structural unit （M,M’=Mo） Fe).
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.
文摘In the study of the catalytic active center of nitrogen fixation in nitrogenase, structural models containing MoFe3S3 or MoFe3S4 cubane-like skeleton have been proposed. The syntheses of these model compounds have since attracted the attention of many chemists. Up to the present time, however, only Holm’s research group has obtained MoFe3S4 single cubane cluster compounds by multi-step cleavage reaction of Mo—Fe—S
文摘老年多脏器功能衰竭（multipleorgan failure in the elderly,MOFE）发病机制复杂,病情凶险,进展迅速,治疗困难,病死率高。现对1998年1月至2008年10月我院480例MOFE患者进行回顾性分析，旨在探讨MOFE发生的相关因素，以期对临床预防和治疗提供参考，报告如下。
