Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site.
Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site.
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DOI：
10.1007/s00775-023-01990-7
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发表时间：
2023-04
影响因子：
3
通讯作者：
Pierce, Brad S. S.
中科院分区：
文献类型：
--
作者：
York, Nicholas J. J.;Lockart, Molly M. M.;Schmittou, Allison N. N.;Pierce, Brad S. S.
硫醇双加氧酶是非血红素单核铁加氧酶的一个子集，可催化含硫醇底物的 O2 依赖性氧化，产生亚磺酸产物。半胱氨酸双加氧酶 (CDO) 和 3-巯基丙酸 (3MPA) 双加氧酶 (MDO) 是该酶家族中特征最广泛的成员。与许多非血红素单核铁氧化酶/加氧酶一样，CDO 和 MDO 在双加氧之前表现出有机底物的专性有序添加。由于这种底物门控 O2 反应性延伸至氧替代物一氧化氮 (NO)，EPR 光谱长期以来一直用于研究[底物:NO:酶]三元复合物。原则上，这些研究可以推断以提供有关分子氧催化转换过程中产生的瞬时铁氧中间体的信息。在这项工作中，我们证明氰化物在有序添加实验中用从固氮杆菌 (AvMDO) 克隆的 MDO 模拟天然硫醇底物。用过量氰化物处理催化活性 Fe(II)-AvMDO 后，添加 NO 产生低自旋 (S = 1/2) (CN/NO)-Fe 络合物。对野生型和 H157N 变体 AvMDO 中产生的该复合物的连续波和脉冲 X 波段 EPR 表征揭示了多个核超精细特征，可诊断酶 Fe 位点的第一和外配位球内的相互作用。经光谱验证的计算模型表明，两个氰化物配体的同时配位取代了 3MPA 的双齿（硫醇和羧酸盐）配位，从而允许在催化相关的 O2 结合位点进行 NO 结合。 AvMDO 与 NO 的这种混杂底物门控反应性与哺乳动物 CDO 对 L-半胱氨酸表现出的高底物特异性提供了有益的对比。
Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for l-cysteine.
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影响因子：
2.9
作者：
Fernandez RL;Elmendorf LD;Smith RW;Bingman CA;Fox BG;Brunold TC
通讯作者：
Brunold TC
DOI：
10.1021/jp404743d
发表时间：
2013-09-12
期刊：
The journal of physical chemistry. B
影响因子：
--
作者：
Casey TM;Grzyska PK;Hausinger RP;McCracken J
通讯作者：
McCracken J
DOI：
10.1016/0926-6593(66)90176-7
发表时间：
1966-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
EWETZ, L;SORBO, B
通讯作者：
SORBO, B
DOI：
10.1139/v97-002
发表时间：
1997-01-01
期刊：
CANADIAN JOURNAL OF CHEMISTRY-REVUE CANADIENNE DE CHIMIE
影响因子：
--
作者：
Forlano, P;Olabe, JA;Blesa, MA
通讯作者：
Blesa, MA
影响因子：
3.9
作者：
Crowell, Joshua K.;Sardar, Sinjinee;Pierce, Brad S.
通讯作者：
Pierce, Brad S.