Brohawn Stephen G, Miksa Irina Rudik, Thorpe Colin
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Biochemistry. 2003 Sep 23;42(37):11074-82. doi: 10.1021/bi0301385.
Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is not a metalloenzyme, and that copper and zinc ions inhibit the oxidation of reduced pancreatic ribonuclease by the enzyme. Studies with Zn(2+), as a redox inactive surrogate for copper, show that one Zn(2+) binds to four-electron-reduced QSOX by diverting electrons away from the flavin and into two of the three redox active disulfide bridges in the enzyme. The resulting zinc complex is modestly air-stable, reverting to a spectrum of the native protein with a t(1/2) of 40 min, whereas the four-electron-reduced native QSOX is reoxidized in less than a second under comparable conditions. Using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), an alternate substrate of QSOX that binds Zn(2+) relatively weakly (unlike dithiothreitol), allows rapid inhibition of oxidase activity to be demonstrated at low micromolar metal levels. Zinc binding was followed by rapid-scanning spectrophotometry. Copper also binds the four-electron-reduced form of QSOX with a visible spectrum suggestive of active site occupancy. In addition to interactions with the reduced enzyme, dialysis experiments show that multiple copper and zinc ions can bind to the oxidized enzyme without the perturbation of the flavin spectrum seen earlier. These data suggest that a reinvestigation of the metal content of skin sulfhydryl oxidases is warranted. The redox-modulated binding of zinc to QSOX is considered in light of evidence for a role of zinc-thiolate interactions in redox signaling and zinc mobilization.
金属和黄素依赖性巯基氧化酶催化二硫键的生成，同时将氧气还原为过氧化氢。据报道，哺乳动物皮肤中的这种酶是铜依赖性的，但最近的蛋白质序列显示它属于Quiescin/巯基氧化酶（QSOX）黄素蛋白家族。这项研究表明，禽类QSOX不是金属酶，铜离子和锌离子会抑制该酶对还原型胰核糖核酸酶的氧化作用。用Zn(2+)（作为铜的氧化还原惰性替代物）进行的研究表明，一个Zn(2+)通过将电子从黄素转移到该酶三个氧化还原活性二硫键中的两个，与四电子还原的QSOX结合。生成的锌复合物在空气中有一定的稳定性，以40分钟的半衰期恢复为天然蛋白质的光谱，而在类似条件下，四电子还原的天然QSOX在不到一秒钟内就会被重新氧化。使用盐酸三(2-羧乙基)膦（TCEP），一种与Zn(2+)结合相对较弱的QSOX替代底物（与二硫苏糖醇不同），可以在低微摩尔金属水平下快速证明氧化酶活性受到抑制。通过快速扫描分光光度法跟踪锌的结合情况。铜也与四电子还原形式的QSOX结合，其可见光谱表明活性位点被占据。除了与还原酶的相互作用外，透析实验表明，多个铜离子和锌离子可以与氧化酶结合，而不会像之前那样扰乱黄素光谱。这些数据表明有必要重新研究皮肤巯基氧化酶的金属含量。鉴于硫醇锌相互作用在氧化还原信号传导和锌动员中的作用证据，对锌与QSOX的氧化还原调节结合进行了探讨。
