Pham D N, Burgess B K
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Biochemistry. 1993 Dec 14;32(49):13725-31. doi: 10.1021/bi00212a043.
Molybdenum nitrogenase is composed of two separately purified proteins designated the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein), with the latter containing the substrate reduction site which is a metal cluster designated the iron-molybdenum cofactor (FeMo cofactor). In addition to its physiological substrates H+ and N2, nitrogenase reduces a number of nonphysiological substrates (e.g. C2H2 and N3-) and interacts with a number of similar molecules (e.g. CH3NC and CO) that serve as specific inhibitors. Despite their great diversity, all substrates are reduced by multiples of two electrons and require equivalent numbers of electrons and protons. Although the electron donor to a substrate is believed to be FeMo cofactor, the nature of the proton donor is unknown and might be different for different substrates. Here we report a three-component buffer assay system that eliminates variables of buffer type, ionic strength, and ATP and reductant availability and that is compatible with the nitrogenase system in the pH range 5.0-9.8. Preincubated studies and studies of the effects of pH on H2 evolution under Ar, H2 evolution under N2, H2 evolution under CO, and C2H2 reduction show that there is a group with a pK of ca. 6.3 that must be deprotonated for substrate reduction to occur and that there is a group with a pK of ca. 9.0 that must be protonated for substrate reduction to occur.(ABSTRACT TRUNCATED AT 250 WORDS)
钼固氮酶由两种分别纯化的蛋白质组成，即铁蛋白（Fe蛋白）和钼铁蛋白（MoFe蛋白），后者含有底物还原位点，该位点是一个金属簇，称为铁钼辅因子（FeMo辅因子）。除了其生理底物H⁺和N₂外，固氮酶还能还原多种非生理底物（如C₂H₂和N₃⁻），并与多种类似分子（如CH₃NC和CO）相互作用，这些分子作为特异性抑制剂。尽管底物种类繁多，但所有底物都是通过两个电子的倍数进行还原的，并且需要等量的电子和质子。虽然底物的电子供体被认为是FeMo辅因子，但质子供体的性质尚不清楚，可能因不同底物而异。在此，我们报道了一种三组分缓冲液测定系统，该系统消除了缓冲液类型、离子强度以及ATP和还原剂可用性的变量，并且在pH范围5.0 - 9.8内与固氮酶系统兼容。预孵育研究以及pH对Ar气氛下H₂释放、N₂气氛下H₂释放、CO气氛下H₂释放和C₂H₂还原影响的研究表明，存在一个pK约为6.3的基团，底物还原发生时该基团必须去质子化，并且存在一个pK约为9.0的基团，底物还原发生时该基团必须质子化。（摘要截于250字）
