Scott D J, Dean D R, Newton W E
Western Regional Research Center, United States Department of Agriculture, Albany, California 94710.
J Biol Chem. 1992 Oct 5;267(28):20002-10.
Unlike wild type, certain Mo-dependent nitrogenases, which are expressed in non-N2-fixing mutant strains of Azotobacter vinelandii and have single amino acid substitutions within a region of the MoFe protein alpha-subunit proposed to encompass an FeMo cofactor-binding domain, are able to catalyze the reduction of acetylene by both two and four electrons to yield ethylene and ethane, respectively (Scott, D. J., May, H. D., Newton, W. E., Brigle, K. E., and Dean, D. R. (1990) Nature 343, 188-190). Although the V-dependent nitrogenase is also able to catalyze the reduction of acetylene to the same two- and four-electron products (Dilworth, M. J., Eady, R. R., Robson, R. L., and Miller, R. W. (1987) Nature 327, 167-168), we find that ethane formation from acetylene catalyzed by the altered Mo-dependent nitrogenases occurs by a different mechanism, which is distinguished by: (i) an increased sensitivity to CO; (ii) the absence of a lag; and (iii) no temperature dependence of product distribution among ethylene and ethane during acetylene reduction. An altered MoFe protein, which was purified from one such mutant strain having the alpha-subunit glutaminyl 191 residue substituted by lysyl, exhibited both a changed S = 3/2 EPR spectrum and changes in the distribution of electrons to various products when compared to wild type. Also, unlike wild type, this altered MoFe protein catalyzed proton reduction that is inhibited by carbon monoxide (CO). Because proton reduction catalyzed by a nitrogenase that has a FeMo cofactor with citrate rather than homocitrate as its organic constituent (Liang, J., Madden, M., Shah, V. K., and Burris, R. H. (1990) Biochemistry 29, 8577-8581) is also inhibited by CO, the possibility arose that changes in the polypeptide environment of FeMo cofactor might have caused a rearrangement in its molecular structure or composition. However, this possibility was ruled out by biochemical reconstitution studies (using FeMo cofactor isolated from both the wild-type and altered MoFe proteins), which were monitored by EPR spectroscopy and resulting catalytic activity.
与野生型不同，某些依赖钼的固氮酶在棕色固氮菌的非固氮突变菌株中表达，并且在钼铁蛋白α亚基的一个区域内有单个氨基酸替换，该区域被认为包含一个铁钼辅因子结合结构域，能够分别通过双电子和四电子催化乙炔还原，生成乙烯和乙烷（斯科特，D. J.，梅，H. D.，牛顿，W. E.，布里格尔，K. E.，和迪恩，D. R.（1990年）《自然》343卷，188 - 190页）。尽管依赖钒的固氮酶也能够催化乙炔还原为相同的双电子和四电子产物（迪尔沃思，M. J.，伊迪，R. R.，罗布森，R. L.，和米勒，R. W.（1987年）《自然》327卷，167 - 168页），但我们发现，由改变后的依赖钼的固氮酶催化乙炔生成乙烷的过程是通过一种不同的机制进行的，其特点是：（i）对一氧化碳的敏感性增加；（ii）没有延迟；（iii）在乙炔还原过程中，乙烯和乙烷之间的产物分布不依赖温度。从一个α亚基谷氨酰胺191残基被赖氨酸取代的突变菌株中纯化得到的一种改变后的钼铁蛋白，与野生型相比，其S = 3/2的电子顺磁共振谱发生了变化，并且向各种产物的电子分布也发生了变化。此外，与野生型不同，这种改变后的钼铁蛋白催化的质子还原受到一氧化碳（CO）的抑制。由于由一种铁钼辅因子以柠檬酸盐而非高柠檬酸盐作为有机成分的固氮酶催化的质子还原（梁，J.，马登，M.，沙阿，V. K.，和伯里斯，R. H.（1990年）《生物化学》2卷，8577 - 8581页）也受到CO的抑制,因此有可能铁钼辅因子多肽环境的变化可能导致了其分子结构或组成的重排。然而，通过生物化学重组研究（使用从野生型和改变后的钼铁蛋白中分离出的铁钼辅因子）排除了这种可能性，这些研究通过电子顺磁共振光谱和产生的催化活性进行监测。
