THE VANADIUM NITROGENASE OF AZOTOBACTER-CHROOCOCCUM - PURIFICATION AND PROPERTIES OF THE VFE PROTEIN
THE VANADIUM NITROGENASE OF AZOTOBACTER-CHROOCOCCUM - PURIFICATION AND PROPERTIES OF THE VFE PROTEIN
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DOI：
10.1042/bj2440197
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发表时间：
1987-05-15
影响因子：
4.1
通讯作者：
HAWKINS, M
中科院分区：
文献类型：
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作者：
EADY, RR;ROBSON, RL;HAWKINS, M
1.将一株缺失常规固氮酶结构基因（nifHDK）的褐球固氮菌的固氮酶活性分为两个组分：含铁蛋白和钒蛋白。2.在SDS存在下，通过10%（w/v）丙烯酰胺凝胶电泳的标准将较大的蛋白纯化至均一。两种类型的亚基（Mr 50，000和55，000）以等量存在。3.该蛋白质的Mr为210，000，每个分子含有2个V原子，23个Fe原子和20个酸不稳定的硫化物基团。Mo含量小于0.06克原子/摩尔。所有常见的氨基酸都存在，以酸性残基为主。超离心分析给出9.7S的最大沉降系数和在5 mg蛋白质时的对称边界。ml-1;解离发生在较低浓度。当在最佳条件下用来自该菌株的互补Fe蛋白测定时，比活性（nmol产物/min每mg蛋白）为1348用于H2释放，350用于NH3形成和608用于乙炔还原。活性是O2不稳定的，在空气中的t1/2为40 s。在低温下，连二亚硫酸盐还原的蛋白质显示e.p.r. g = 5.6、4.35、3.77和1.93处的信号，与S = 3/2基态一致，具有额外的S = 1/2中心，从而产生g = 1.93处的特征。连二亚硫酸盐还原和硫堇氧化蛋白的紫外光谱非常相似。氧化导致可见光区吸光度普遍增加。还原蛋白光谱中380 nm处的肩峰被氧化后330 nm和420 nm附近的肩峰取代。
1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes for conventional nitrogenase (nifHDK) was separated into two components: an Fe-containing protein and a vanadoprotein. 2. The larger protein was purified to homogeneity by the criterion of electrophoresis of 10% (w/v) acrylamide gels in the presence of SDS. Two types of subunit, of Mr 50,000 and 55,000, were present in equal amounts. 3. The protein had an Mr of 210,000 and contained 2 V atoms, 23 Fe atoms and 20 acid-labile sulphide groups per molecule. The Mo content was less than 0.06 g-atom/mol. All the common amino acids were present, with a predominance of acidic residues. Ultracentrifugal analysis gave a maximum sedimentation coefficient of 9.7 S and a symmetrical boundary at 5 mg of protein .cntdot. ml-1; dissociation occurred at lower concentrations. The specific activities (nmol of product/min per mg of protein), when assayed under optimum conditions with the complementary Fe protein from this strain, were 1348 for H2 evolution, 350 for NH3 formation and 608 for acetylene reduction. Activity was O2-labile, with a t1/2 of 40 s in air. At low temperatures the dithionite-reduced protein showed e.p.r. signals at g = 5.6, 4.35, 3.77 and 1.93, consistent with an S = 3/2 ground state with an additional S = 1/2 centre giving rise to the feature at g = 1.93. The u.v. spectra of dithionite-reduced and thionine-oxidized protein were very similar. Oxidation resulted in a general increase in absorbance in the visible region. The shoulder at 380 nm in the spectrum of reduced protein was replaced with shoulders near 330 nm and 420 nm on oxidation.