Spectroscopic characterization of the NO adduct of hydroxylamine oxidoreductase.
Spectroscopic characterization of the NO adduct of hydroxylamine oxidoreductase.
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DOI：
10.1021/bi011332z
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发表时间：
2002-03
期刊：
影响因子：
2.9
通讯作者：
M. Hendrich;A. Upadhyay;J. Riga;D. Arciero;A. Hooper
中科院分区：
文献类型：
--
作者：
M. Hendrich;A. Upadhyay;J. Riga;D. Arciero;A. Hooper
来自自养硝化细菌欧洲亚硝化细菌的羟胺氧化还原酶 (HAO) 催化 NH2OH 氧化为 NO2-。该酶每个亚基含有八个血红素，参与催化和电子传输。研究发现，NO 与酶结合并抑制电子流向受体蛋白细胞色素 c554。发现 NO 可以部分或完全氧化还原的 HAO，但 NO 不会还原三价 HAO。由于 NO 可以被 HAO 还原但不能氧化成产物，因此 NO 不被认为是催化机制中的长寿命中间体。底物氧化发生在存在结合的 NO 或氰化物的情况下，这表明底物与 HAO 存在第二个相互作用位点，并提供了恢复 NO 抑制形式的酶的方法。将NO添加到氧化的HAO中后，来自活性位点的整数自旋EPR信号消失，来自NO的红外谱带出现在1920 cm(-1)处，并且在穆斯堡尔谱中出现反磁性四极铁双峰，其δ = 0.06 mm/s和DeltaEq = 2.1 mm/s。 NO 伸缩频率和穆斯堡尔参数是 [FeNO]6 血红素复合物的特征。还提供了有关铁肌红蛋白-NO 的新穆斯堡尔数据以供比较。结果表明，NO 与血红素 P460 结合，并且整数自旋 EPR 信号的丢失是由于血红素 P460 转换为抗磁性 S = 0 状态，并伴随着与邻近血红素 6 的磁相互作用的丧失。在以前的研究中，血红素 P460-血红素 6 相互作用受到底物或氰化物结合的影响，因此无法观察到血红素 6 的信号。相反，在这项工作中，NO 引起的信号丢失伴随着先前未观察到的来自血红素 6 的大 g(max)（或 HALS）低自旋 EPR 信号的出现。
Hydroxylamine oxidoreductase (HAO) from the autotrophic nitrifying bacterium Nitrosomonas europaea catalyzes the oxidation of NH2OH to NO2-. The enzyme contains eight hemes per subunit which participate in catalysis and electron transport. NO is found to bind to the enzyme and inhibit electron flow to the acceptor protein, cytochrome c554. NO is found to oxidize either partially or fully reduced HAO, but NO will not reduce ferric HAO. Since NO can be reduced but not oxidized to product by HAO, NO is not considered to be a long-lived intermediate in the catalytic mechanism. Substrate oxidation occurs in the presence of bound NO or cyanide, suggesting a second interaction site for substrate with HAO and providing a means for recovery of the NO-inhibited form of the enzyme. Upon addition of NO to oxidized HAO, the integer-spin EPR signal from the active site vanishes, an IR band from NO appears at 1920 cm(-1), and a diamagnetic quadrupole iron doublet appears in Mössbauer spectroscopy with delta = 0.06 mm/s and DeltaEq = 2.1 mm/s. The NO stretching frequency and Mössbauer parameters are characteristic of an [FeNO]6 heme complex. New Mössbauer data on ferric myoglobin-NO are also presented for comparison. The results indicate that NO binds to heme P460 and that the loss of the integer-spin EPR signal is due to the conversion of heme P460 to a diamagnetic S = 0 state and concomitant loss of magnetic interaction with neighboring heme 6. In previous studies where the heme P460-heme 6 interaction was affected by substrate or cyanide binding, a signal attributable to heme 6 was not observable. In contrast, in this work, the NO-induced loss of the signal is accompanied by the appearance of a previously unobserved large g(max) (or HALS) low-spin EPR signal from heme 6.