Yousafzai F K, Eady R R
Nitrogen Fixation Laboratory, John Innes Centre, Colney, Norwich NR4 7UH, UK.
Biochem J. 1999 May 1;339 ( Pt 3)(Pt 3):511-5.
When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4(-) in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S=3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4 K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4(-) was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution.
当肺炎克雷伯菌固氮酶的MoFe（Kp1）和Fe（Kp2）组分蛋白在连二亚硫酸盐作为还原剂存在的情况下与MgADP和AlF4(-)一起温育时，会产生一种稳定的假定过渡态复合物[尤萨夫扎伊和伊迪（1997年）《生物化学杂志》326卷，637 - 640页]。令人惊讶的是，与还原型Kp2相关的电子顺磁共振（EPR）信号无法检测到，但Kp1保留了由该蛋白的MoFe辅因子中心的连二亚硫酸盐还原态产生的S = 3/2 EPR信号。这与复合物中Fe蛋白的[Fe4S4]中心被氧化是一致的，并且在棕色固氮菌的复合物中也有类似的观察结果[斯皮、阿伦德森、瓦辛克、马里特、哈根和哈克（1998年）《欧洲生物化学学会联合会快报》432卷，55 - 58页]。对于Kp2失去的电子的去向，尚未有令人满意的解释。然而，我们在此报告，在氩气氛围下制备肺炎克雷伯菌的MgADP - AlF4复合物的过程中，会释放出与Kp1的铁钼辅因子（FeMoco）含量的一半相对应量的H2（FeMoco是固氮酶可能的催化位点，其组成为Mo:Fe7:S9:高柠檬酸）。这很令人惊讶，因为在不存在通常被认为是固氮酶功能所必需的MgATP且存在作为固氮酶强竞争性抑制剂的MgADP的温育过程中观察到了活性。在含有MgADP但不含MgATP的反应混合物中，也观察到了在不存在AlF4(-)的情况下固氮酶形成H2的现象。当用越来越多的Kp2滴定Kp1时，该反应呈现出饱和动力学，并且在饱和时，形成的H2量与Kp1的FeMoco含量呈化学计量关系。H2形成速率对[MgADP]的依赖性与MgATP污染导致的活性不一致。我们得出结论，MgATP对于固氮酶还原H+并非必不可少，因为MgADP支持非常低的析氢速率。
