Schneider K, Gollan U, Dröttboom M, Selsemeier-Voigt S, Müller A
Lehrstuhl für Anorganische Chemie I, Fakultät für Chemie der Universität Bielefeld, Germany.
Eur J Biochem. 1997 Mar 15;244(3):789-800. doi: 10.1111/j.1432-1033.1997.t01-1-00789.x.
The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (delta nifHDK, delta modABCD strain) and purified in a one-day procedure that included only one column-chromatography step (DEAE-Sephacel). This procedure yielded component 1 (FeFe protein, Rc1Fe), which was more than 95% pure, and an approximately 80% pure component 2 (Fe protein, Rc2Fe). The highest specific activities, which were achieved at an Rc2Fe/Rc1Fe molar ratio of 40:1, were 260 (C2H4 from C2H2), 350 (NH3 formation), and 2400 (H2 evolution) nmol product formed x min(-1) x mg protein(-1). The purified FeFe protein contained 26 +/- 4 Fe atoms; it did not contain Mo, V, or any other heterometal atom. The most significant catalytic property of the iron-only nitrogenase is its high H2-producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N2 reduction, the activity ratios (mol N2 reduced/mol H2 produced) obtained were 1:1 (molybdenum nitrogenase) and 1:7.5 (iron nitrogenase). The Rc1Fe protein has only a very low affinity for C2H2. The Km value determined (12.5 kPa), was about ninefold higher than the Km for Rc1Mo (1.4 kPa). The proportion of ethane produced from acetylene (catalyzed by the iron nitrogenase), was strictly pH dependent. It corresponded to 5.5% of the amount of ethylene at pH 6.5 and was almost zero at pH values greater than 8.5. In complementation experiments, component 1 proteins coupled very poorly with the 'wrong' component 2. Rc1Fe, if complemented with Rc2Mo, showed only 10-15% of the maximally possible activity. Cross-reaction experiments with isolated polyclonal antibodies revealed that Rc1Fe and Rc1Mo are immunologically not related. The most active Rc1Fe samples appeared to be EPR-silent in the Na2S2O4-reduced state. However, on partial oxidation with K3[Fe(CN)6] or thionine several signals occurred. The most significant signal appears to be the one at g = 2.27 and 2.06 which deviates from all signals so far described for P clusters. It is a transient signal that appears and disappears reversibly in a redox potential region between -100 mV and +150 mV. Another novel EPR signal (g = 1.96, 1.92, 1.77) occurred on further reduction of Rc1Fe by using turnover conditions in the presence of a substrate (N2, C2H2, H+).
仅含铁固氮酶的组成蛋白是从荚膜红细菌（ΔnifHDK，ΔmodABCD菌株）中分离出来的，并通过一个仅包含一步柱色谱步骤（DEAE-琼脂糖凝胶）的一日程序进行纯化。该程序得到了纯度超过95%的组分1（铁铁蛋白，Rc1Fe）和纯度约为80%的组分2（铁蛋白，Rc2Fe）。在Rc2Fe/Rc1Fe摩尔比为40:1时达到的最高比活性分别为：260（由乙炔生成乙烯）、350（氨的形成）和2400（氢气释放）nmol产物形成·min⁻¹·mg蛋白⁻¹。纯化的铁铁蛋白含有26±4个铁原子；它不含钼、钒或任何其他异金属原子。仅含铁固氮酶最显著的催化特性是其高氢气产生活性，与传统钼固氮酶的活性相比，它受竞争性底物的抑制要小得多。在氮气还原的最佳条件下，得到的活性比（还原的氮气摩尔数/产生的氢气摩尔数）分别为1:1（钼固氮酶）和1:7.5（铁固氮酶）。Rc1Fe蛋白对乙炔的亲和力非常低。测定的Km值（12.5 kPa）比Rc1Mo的Km值（1.4 kPa）高约9倍。由乙炔催化生成的乙烷比例严格依赖于pH值。在pH 6.5时，它相当于乙烯量的5.5%，在pH值大于8.5时几乎为零。在互补实验中，组分1蛋白与“错误”的组分2的偶联非常差。如果用Rc2Mo进行互补，Rc1Fe仅显示出最大可能活性的10 - 15%。用分离的多克隆抗体进行的交叉反应实验表明，Rc1Fe和Rc1Mo在免疫学上不相关。最具活性的Rc1Fe样品在连二亚硫酸钠还原状态下似乎是EPR沉默的。然而，在用铁氰化钾或亚甲蓝进行部分氧化时会出现几个信号。最显著的信号似乎是在g = 2.27和2.06处的信号，它与迄今为止描述的所有P簇信号都不同。这是一个瞬态信号，在-100 mV至+150 mV的氧化还原电位区域中可逆地出现和消失。在存在底物（N₂、C₂H₂、H⁺）使用周转条件进一步还原Rc1Fe时，出现了另一个新的EPR信号（g = 1.96、1.92、1.77）。
