Duyvis M G, Wassink H, Haaker H
Laboratory of Biochemistry, Department of Biomolecular Sciences, Wageningen Agricultural University, The Netherlands.
Biochemistry. 1998 Dec 15;37(50):17345-54. doi: 10.1021/bi981509y.
Nitrogenase consists of two metalloproteins (Fe protein and MoFe protein) which are assumed to associate and dissociate to transfer a single electron to the substrates. This cycle, called the Fe protein cycle, is driven by MgATP hydrolysis and is repeated until the substrates are completely reduced. The rate-limiting step of the cycle, and substrate reduction, is suggested to be the dissociation of the Fe protein-MoFe protein complex which is obligatory for the reduction of the Fe protein [Thorneley, R. N. F., and Lowe, D. J. (1983) Biochem. J. 215, 393-403]. This hypothesis is based on experiments with dithionite as the reductant. We also tested besides dithionite flavodoxin hydroquinone, a physiological reductant. Two models could describe the experimental data of the reduction by dithionite. The first model, with no reduction of Fe protein bound to MoFe protein, predicts a rate of dissociation of the protein complex of 8.1 s-1. This rate is too high to be the rate-limiting step of the Fe protein cycle (kobs = 3.0 s-1). The second model, with reduction of the Fe protein in the nitrogenase complex, predicts a rate of dissociation of the protein complex of 2.3 s-1, which in combination with reduction of the nitrogenase complex can account for the observed turnover rate of the Fe protein cycle. When flavodoxin hydroquinone (155 microM) was the reductant, the rate of reduction of oxidized Fe protein in the nitrogenase complex (kobs approximately 400 s-1) was 100 times faster than the turnover rate of the cycle with flavodoxin as the reductant (4 s-1). Pre-steady-state electron uptake experiments from flavodoxin hydroquinone indicate that before and after reduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Fe protein cycle. These results are discussed in the context of the kinetic models of the Fe protein cycle of nitrogenase.
固氮酶由两种金属蛋白（铁蛋白和钼铁蛋白）组成，它们被认为会结合和解离，将单个电子传递给底物。这个循环，称为铁蛋白循环，由MgATP水解驱动，并重复进行，直到底物被完全还原。该循环以及底物还原的限速步骤被认为是铁蛋白 - 钼铁蛋白复合物的解离，这对于铁蛋白的还原是必不可少的[索恩利，R. N. F.，和洛威，D. J.（1983年）《生物化学杂志》215卷，393 - 403页]。这个假设是基于以连二亚硫酸盐作为还原剂的实验。除了连二亚硫酸盐，我们还测试了黄素氧还蛋白对苯二酚，一种生理性还原剂。有两种模型可以描述连二亚硫酸盐还原的实验数据。第一个模型，即与钼铁蛋白结合的铁蛋白不被还原，预测蛋白复合物的解离速率为8.1 s⁻¹。这个速率太高，不可能是铁蛋白循环的限速步骤（观测到的速率常数kobs = 3.0 s⁻¹）。第二个模型，即固氮酶复合物中的铁蛋白被还原，预测蛋白复合物的解离速率为2.3 s⁻¹，这与固氮酶复合物的还原相结合，可以解释观测到的铁蛋白循环的周转速率。当黄素氧还蛋白对苯二酚（155微摩尔）作为还原剂时，固氮酶复合物中氧化态铁蛋白的还原速率（kobs约为400 s⁻¹）比以黄素氧还蛋白作为还原剂时循环的周转速率（4 s⁻¹）快100倍。来自黄素氧还蛋白对苯二酚的稳态前电子摄取实验表明，在固氮酶复合物还原之前和之后都会发生相对缓慢的反应，这限制了铁蛋白循环的速率。这些结果在固氮酶铁蛋白循环的动力学模型背景下进行了讨论。
