Characterization of a nitro-genase CrFe protein from a mutant UW3 of Azotobacter vinelandii grown on a Cr-containing medium

通过 DEAE-52 的列上的缺氧的层析， Q-Sepharose andSeph 压克力 S-200， CrFe 蛋白质准备从 UW3 被获得， Azotobactervinelandii 的异种，它在包含 Cr 媒介上成长了很好。与 MoFe 蛋白质(OP Av1 ) 相比 fromwild 类型紧张 OP，在准备的约 50% 蛋白质有类似的子单元作文并且与 OP Av1 的抗体有有免疫力的反应。准备有 C_2H_2- H~+-andN_2 的 -40 百分比(在 H~+ 减小活动在之间由差别表示了在 Ar 下面并且在 N_2 下面) 到那些的 OP Av1 和 OP Av1 的类似的电子对的减小活动。并且金属分析证明准备包含了 Fe， Cr 和瞬间。，在在约 450 nm 的准备的圆形的二色性(CD ) 光谱类似于 OP Av1 的出现在一样的 g 价值的 threeEPR 信号的相对紧张(g 约 4.3， 3.7 和 2.0 ) 是不同的。EPR-basedcalculated 结果显示出那(1 ) 到 Cr 的瞬间并且 Fe 到的比率(Cr+Mo ) 分别地， CrFe 蛋白质准备， 0.41 和 15 正在显示在准备，到 MoFe 蛋白质的比率包含 ofCr 蛋白质是大约 2.5；(2 ) 到 Fe 并且到准备的 Cr 的活动的比率分别地接近了活动的比率到 Fe 并且到 OP Av1 的瞬间；(3 ) EPR 的比率在到 CrFe 蛋白质的 Cr 的值大约到瞬间的约 83% 那些， 0% 和约 40% 的上述三 g 表明紧张。OP Av1，分别地。结果进一步显示 CrFe 蛋白质力量是一个新 ni-trogenase 部件我有在余因子由 Mn MoFe 蛋白质包括 metallocluster 到那些而不是瞬间的简单代替的类似的功能和结构的蛋白质。

Characterization of a FeMo cofactor-deficient MoFe protein from a nifE-deleted strain(DJ35)of Azotobacter vinelandii

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.