Protein oxidation involved in Cys-Tyr post-translational modification
Protein oxidation involved in Cys-Tyr post-translational modification
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DOI：
10.1016/j.jinorgbio.2017.08.028
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发表时间：
2017-11-01
影响因子：
3.9
通讯作者：
Benson, David E.
中科院分区：
文献类型：
--
作者：
Hromada, Susan E.;Hilbrands, Adam M.;Benson, David E.
一些后修饰的酪氨酸可以进行类似于金属辅因子的可逆氧化还原化学。这些酪氨酸修饰中研究最多的是半乳糖氧化酶中的分子内硫醚交联的3'-（S-半胱氨酰）酪氨酸（Cys-Tyr）。这种铜介导的酪氨酸修饰半乳糖氧化酶涉及直接电子转移（内球）的协调酪氨酸。哺乳动物半胱氨酸双加氧酶还含有Cys-Tyr，其可能通过从远离母体残基约6埃的非血红素铁中心的外层电子转移形成。孤儿蛋白（BF 4112），服从紫外光谱表征，也已被证明形成Cys-Tyr之间的Tyr 52和Cys 98通过相邻的Cu2+离子负载，单核金属离子结合位点。变性条件下的天然Cys-Tyr荧光为Cys-Tyr产率测定提供了更稳健的方法。在该荧光测定中，通过氯化胍提供了相对于3，3'-二酪氨酸的Cys-Tyr特异性。用Phe替代Tyr 52或用Zn 2+离子替代Cue离子消除了Cys-Tyr形成。通过表面Tyr突变为Phe（Y4F/Y109F，50%）和Cys 98突变为Ser（25%），基于Cys-Tyr荧光的产率降低但未完全去除。C98S BF 4112的小吸光度和荧光发射强度是令人惊讶的，直到观察到显著红移的发射。通过还原变性SDS-PAGE观察到的红移发射光谱和单体到二聚体的位移证明了当Cys 98被Ser取代时表面酪氨酸基产物（二酪氨酸）。这些结果证明了在Cys-Tyr形成期间BF 4112中的表面酪氨酸氧化，并且蛋白质氧化可以是形成蛋白质衍生辅因子的显著副反应。
Some post-translationally modified tyrosines can perform reversible redox chemistry similar to metal cofactors. The most studied of these tyrosine modifications is the intramolecular thioether-crosslinked 3'-(S-cysteinyl)tyrosine (Cys-Tyr) in galactose oxidase. This Cu-mediated tyrosine modification in galactose oxidase involves direct electron transfer (inner-sphere) to the coordinated tyrosine. Mammalian cysteine dioxygenase enzymes also contain a Cys-Tyr that is formed, presumably, through outer-sphere electron transfer from a non-heme iron center -6 angstrom away from the parent residues. An orphan protein (BF4112), amenable to UV spectroscopic characterization, has also been shown to form Cys-Tyr between Tyr 52 and Cys 98 by an adjacent Cu2+ ion loaded, mononuclear metal ion binding site. Native Cys-Tyr fluorescence under denaturing conditions provides a more robust methodology for Cys-Tyr yield determination. Cys-Tyr specificity, relative to 3,3'-dityrosine, was provided in this fluorescence assay by guanidinium chloride. Replacing Tyr 52 with Phe or the Cue ion with a Zn2+ ion abolished Cys-Tyr formation. The Cys-Tyr fluorescence-based yields were decreased but not completely removed by surface Tyr mutations to Phe (Y4F/Y109F, 50%) and Cys 98 to Ser (25%). The small absorbance and fluorescence emission intensities for C98S BF4112 were surprising until a significantly red-shifted emission was observed. The red-shifted emission spectrum and monomer to dimer shift seen by reducing, denaturing SDS-PAGE demonstrate a surface tyrosyl radical product (dityrosine) when Cys 98 is replaced with Ser. These results demonstrate surface tyrosine oxidation in BF4112 during Cys-Tyr formation and that protein oxidation can be a significant side reaction in forming protein derived cofactors.