Correlation of optical and EPR signals with the P460 heme of hydroxylamine oxidoreductase from Nitrosomonas europaea.
Correlation of optical and EPR signals with the P460 heme of hydroxylamine oxidoreductase from Nitrosomonas europaea.
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光学和 EPR 信号与欧洲亚硝化单胞菌羟胺氧化还原酶 P460 血红素的相关性。
DOI：
10.1021/bi972187l
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Hooper,AB
中科院分区：
文献类型：
--
作者：
Arciero,DM;Golombek,A;Hendrich,MP;Hooper,AB
欧洲亚硝化单胞菌的羟胺氧化还原酶（HAO）催化NH2OH四电子氧化为NO2-。三聚体酶的每个亚基含有七个血红素和一个血红素 P460。在之前的工作中 [Hendrich, M. P., et al. (1994)J.Am.Chem.Soc.116, 11961−11968]，从静止酶的活性位点发现了整数自旋 EPR 信号 atg= 7.7。这个新信号被分配给包含铁血红素 P460 和铁血红素的交换耦合簇。本文介绍了 HAO 的电化学滴定，其中监测 EPR 信号和光带（据信与 P460 血红素相关）。在EPR滴定中，随着Em8=-140mV的氧化还原中心还原，整数自旋信号消失。然后，当Em8 = -190 mV的氧化还原中心还原时，出现ag = 6型信号，该信号先前已被分配给HAO的铁P460血红素的高自旋形式。然而，在-140至-190 mV范围内，我们无法识别出归因于P460中心的额外EPR信号。因此，在滴定实验中，HAO 的氧化 P460 血红素的电子环境在还原之前似乎经历了三个状态，其中一个中间状态不易被 X 波段 EPR 检测到。 P460 血红素的 c-血红素伴侣的最佳候选者是 -190 mV 的血红素，它对应于晶体结构的血红素 6。交换耦合血红素簇的一个可能的功能是促进底物的双电子氧化步骤。 HAO 的早期分光电位滴定 [Collins, M. J., et al. (1993)J.Biol.Chem.268, 14655−14662] 识别出一条宽而弱的光学带，中心位于 740 nm 附近，暂时归属于氧化的 P460 血红素。通过在几个 pH 值下进行额外的分光电位滴定以及通过连二亚硫酸盐还原 HAO 后的快速动力学实验，这一任务得到了加强。在完全氧化的 HAO 中未观察到 740 nm 波段。在分光电位滴定中，它的出现不能与特定血红素的还原相关，也不能模拟能斯特单电子氧化还原中心。相反，740 nm 波段存在的电势范围取决于滴定是在氧化方向还是还原方向进行。一种可能的解释是，740 nm 谱带是氧化高自旋 P460 血红素的特性，而不是低自旋态的特性，并且两种自旋态之间的转变发生在不同的电位，具体取决于电化学滴定的方向。
Hydroxylamine oxidoreductase (HAO) ofNitrosomonas europaeacatalyzes the four-electron oxidation of NH2OH to NO2-. Each subunit of the trimeric enzyme contains sevenc-hemes and one heme P460. In previous work [Hendrich, M. P., et al. (1994)J.Am.Chem.Soc.116, 11961−11968], an integer-spin EPR signal atg= 7.7 was discovered from the active site of the resting enzyme. This new signal was assigned to an exchange-coupled cluster containing ferric heme P460 and a ferricc-heme. An electrochemical titration of HAO is presented here in which EPR signals and optical bands, believed to be associated with the P460 heme, are monitored. In the EPR titration, as a redox center withEm8= −140 mV becomes reduced, the integer-spin signal disappears. Then, upon reduction of a redox center withEm8= −190 mV, ag= 6 type signal, which has been previously assigned to a high-spin form of the ferric P460 heme of HAO, appears. However, in the −140 to −190 mV range, we have been unable to identify an additional EPR signal attributable to the P460 center. Thus, the electronic environment of oxidized P460 heme of HAO appears to pass through three states before reduction in a titration experiment, with an intermediate state that is not readily detectable by X-band EPR. The best candidate for thec-heme partner of the P460 heme is the heme at −190 mV, which would correspond to heme 6 of the crystal structure. A possible function of the exchange-coupled heme cluster is to facilitate two-electron oxidation steps of the substrate. An earlier spectropotentiometric titration of HAO [Collins, M. J., et al. (1993)J.Biol.Chem.268, 14655−14662] identified a broad, weak optical band, centered near 740 nm, that was tentatively assigned to the oxidized P460 heme. This assignment has been strengthened by additional spectropotentiometric titrations at several values of pH and also by rapid kinetic experiments following the reduction of HAO by dithionite. The 740 nm band is not observed in fully oxidized HAO. In the spectropotentiometric titrations, its appearance cannot be correlated with reduction of a specificc-heme nor modeled to a Nernstian one-electron redox center. Instead, the range of potential in which the 740 nm band is present depends on whether the titration is carried out in an oxidative or reductive direction. One possible interpretation is that the 740 nm band is a property of the oxidized high-spin P460 heme but not of the low-spin state, and that the transition between the two spin states occurs at different potentials depending on the direction of the electrochemical titration.