An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide
An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide
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DOI：
10.1074/jbc.272.33.20340
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发表时间：
1997-08-15
影响因子：
4.8
通讯作者：
Beinert, H
中科院分区：
文献类型：
--
作者：
Kennedy, MC;Antholine, WE;Beinert, H
细胞研究表明，某些Fe-S蛋白，特别是乌头酸酶，是一氧化氮的靶标，具体地说，NO参与了含有[4Fe-4S]簇的胞浆乌头酸酶活性转化为其脱氧型铁调节蛋白的细胞内过程。我们通过跟踪酶活性和观察原始反应物没有显示的EPR信号的形成，体外研究了NO与线粒体和胞质乌头酸酶纯化形式的反应。在厌氧条件下，这两种同工酶都被NO溶液或产生NO的NONOates灭活，在底物存在或不存在的情况下，伴随着[3Fe-4S]团簇的g=2.02信号和d(7)状态下蛋白质结合的二亚硝基-铁-二硫醇络合物的g=2.04信号的出现，此外，在胞浆乌头酸酶的反应中，观察到硫基自由基的瞬时形成，g(平行)=2.11，g(垂直)=2.03，在NO的厌氧加成过程中，酶的[3Fe-4S]簇的解体也伴随着大约2.04种的g的形成，在线粒体乌头酸酶的情况下，出现了大约2.032的瞬时信号atg。这个信号暂时被认为是线粒体蛋白质的铁-亚硝基复合体的d(9)形式。用超氧阴离子或过氧亚硝酸根使两种乌头酸酶的[4Fe-4S]形式失活，产生g=2.02[3Fe-4S]蛋白质。
Cellular studies have indicated that some Fe-S proteins, and the aconitases in particular, are targets for nitric oxide, Specifically, NO has been implicated in the intracellular process of the conversion of active cytosolic aconitase containing a [4Fe-4S] cluster, to its apo-form which functions as an iron-regulatory protein, We have undertaken the in vitro study of the reaction of NO with purified forms of both mitochondrial and cytosolic aconitases by following enzyme activity and by observing the formation of EPR signals not shown by the original reactants, Inactivation by either NO solutions or NO-producing NONOates under anaerobic conditions is seen for both enzyme isoforms, This inactivation, which occurs in the presence or absence of substrate, is accompanied by the appearance of the g = 2.02 signals of the [3Fe-4S] clusters and the g approximate to 2.04 signal of a protein-bound dinitrosyl-iron-dithiol complex in the d(7) state, In addition, in the reaction of cytosolic aconitase, the transient formation of a thiyl radical, g(parallel to) = 2.11 and g(perpendicular to) = 2.03, is observed, Disassembly of the [3Fe-4S] clusters of the inactive forms of the enzymes upon the anaerobic addition of NO is also accompanied by the formation of the g approximate to 2.04 species and in the case of mitochondrial aconitase, a transient signal atg approximate to 2.032 appeared. This signal is tentatively assigned to the d(9) form of an iron-nitrosylhistidyl complex of the mitochondrial protein. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g = 2.02 [3Fe-4S] proteins.