Isolation and characterization of an acetylene-resistant nitrogenase
Isolation and characterization of an acetylene-resistant nitrogenase
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DOI：
10.1074/jbc.275.15.11459
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发表时间：
2000-04-14
影响因子：
4.8
通讯作者：
Dean, DR
中科院分区：
文献类型：
--
作者：
Christiansen, J;Cash, VL;Dean, DR
研究了一种分离棕色固氮菌突变株的遗传策略，该突变株表现出对乙炔抑制的体内固氮酶活性抗性，检测了该菌株产生的改变的固氮酶MoFe蛋白的动力学特征，该蛋白的α-亚基Gly（69）残基被丝氨酸取代，与其他研究一致，表明MoFe蛋白通常含有至少两个乙炔结合/还原位点。其中第一个是高亲和力位点，并且是典型的乙炔还原测定期间主要访问的位点。本工作的结果表明，这个乙炔结合/还原网站是不直接相关的氮还原的机制，因为它可以被消除或严重改变，而不会显着影响氮还原。该位点的消除还导致表现出低亲和力乙炔结合位点，乙炔和氮都能够以大致相同的亲和力结合到该低亲和力乙炔结合位点。与正常酶相反，氮和乙炔结合到改变的MoFe蛋白是相互竞争的。α-Ser（69）取代的位置被解释为表明由Cu-亚基瓦尔（70）残基封端的FeMo辅因子的4Fe-4S面是FeMo辅因子内乙炔以高亲和力结合的最可能区域。
A genetic strategy was developed for the isolation of a mutant strain of Azotobacter vinelandii that exhibits in vivo nitrogenase activity resistant to inhibition by acetylene, Examination of the kinetic features of the altered nitrogenase MoFe protein produced by this strain, which has serine substituted for the alpha-subunit Gly(69) residue, is consistent with other studies that indicate the MoFe protein normally contains at least two acetylene binding/reduction sites. The first of these is a high affinity site and is the one primarily accessed during typical acetylene reduction assays. Results of the present work indicate that this acetylene binding/reduction site is not directly relevant to the mechanism of nitrogen reduction because it can be eliminated or severely altered without significantly affecting nitrogen reduction. Elimination of this site also results in the manifestation of a low affinity acetylene-binding site to which both acetylene and nitrogen are able to bind with approximately the same affinity. In contrast to the normal enzyme, nitrogen and acetylene binding to the altered MoFe protein are mutually competitive. The location of the alpha-Ser(69) substitution is interpreted to indicate that the 4Fe-4S face of the FeMo cofactor capped by the cu-subunit Val(70) residue is the most likely region within FeMo cofactor to which acetylene binds with high affinity.