Addition of an external electron donor to in vitro assays of cysteine dioxygenase precludes the need for exogenous iron
Addition of an external electron donor to in vitro assays of cysteine dioxygenase precludes the need for exogenous iron
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DOI：
10.1016/j.abb.2012.03.006
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发表时间：
2012-05-01
影响因子：
3.9
通讯作者：
Ellis, Holly R.
中科院分区：
文献类型：
--
作者：
Imsand, Erin M.;Njeri, Catherine W.;Ellis, Holly R.
半胱氨酸双加氧酶 (CDO) 利用 3-His 面部三联体来协调其金属中心。细胞裂解物中存在的重组 CDO 主要以亚铁形式存在，并表现出显着的催化活性。在纯化过程中从还原性细胞环境中去除 COO 会导致结合铁的损失和 99% 以上的剩余金属中心的氧化。分离的重组酶具有与 L-半胱氨酸氧化背景水平相当的活性，证实 CDO 在催化所需的有氧条件下没有活性。在测定中加入外源亚铁会导致非酶产物形成；然而，在纯化蛋白质的测定中添加外部还原剂导致 COO 活性恢复。在还原剂存在下，CDO 的 EPR 光谱证实恢复的活性与铁还原成亚铁形式一致。在 L-半胱氨酸存在下分离的酶几乎不与双氧类似物反应，但当与外部还原剂预孵育时亲和力增加。这些研究揭示了已报道的 CDO 动力学参数之间的差异，并且还比较了 3-His 和 2-His/1-羧酸亚铁酶在双氧存在下的稳定性。 (C) 2012 Elsevier Inc. 保留所有权利。
Cysteine dioxygenase (CDO) utilizes a 3-His facial triad for coordination of its metal center. Recombinant CDO present in cellular lysate exists primarily in the ferrous form and exhibits significant catalytic activity. Removal of COO from the reducing cellular environment during purification results in the loss of bound iron and oxidation of greater than 99% of the remaining metal centers. The as-isolated recombinant enzyme has comparable activity as the background level of L-cysteine oxidation confirming that CDO is inactive under the aerobic conditions required for catalysis. Including exogenous ferrous iron in assays resulted in non-enzymatic product formation; however, addition of an external reductant in assays of the purified protein resulted in the recovery of COO activity. EPR spectroscopy of CDO in the presence of a reductant confirms that the recovered activity is consistent with reduction of iron to the ferrous form. The as-isolated enzyme in the presence of L-cysteine was nearly unreactive with the dioxygen analog, but had increased affinity when pre-incubated with an external reductant. These studies shed light on the discrepancies among reported kinetic parameters for CDO and also juxtapose the stability of the 3-His and 2-His/1-carboxylate ferrous enzymes in the presence of dioxygen. (C) 2012 Elsevier Inc. All rights reserved.