Modification by homocysteine thiolactone affects redox status of cytochrome c
Modification by homocysteine thiolactone affects redox status of cytochrome c
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DOI：
10.1021/bi602463m
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发表时间：
2007-05-29
期刊：
影响因子：
2.9
通讯作者：
Jakubowski, Hieronim
中科院分区：
文献类型：
--
作者：
Perla-Kajan, Joanna;Marczak, Lukasz;Jakubowski, Hieronim
同型半胱氨酸 (Hcy)-硫内酯介导 Hcy 翻译后掺入人类蛋白质中。蛋白质 N-同型半胱氨酸化对蛋白质结构和功能有害，并且与在人类和实验动物中观察到的高同型半胱氨酸血症的病理生理学有关。 Hcy-硫内酯的修饰可能会通过影响必需赖氨酸残基的功能而直接产生有害作用，或通过干扰其他必需残基或辅因子的功能而间接产生有害作用。先前的工作表明，细胞色素 c 对 Hcy-硫内酯非常敏感，这会导致 N-Hcy-细胞色素 c 多聚体的形成。然而，目前尚不清楚细胞色素c中的哪些位点容易附着Hcy，以及N-连接的Hcy是否会影响细胞色素c的结构和氧化还原功能。在这里，我们发现细胞色素 c 的 4 个赖氨酸残基（Lys8 或 -13、Lys86 或 -87、Lys99 和 Lys100）对 N-同型半胱氨酸化敏感。我们还表明，1 mol 赖氨酸/mol 蛋白质的 N-同型半胱氨酸化通过使其还原而影响细胞色素 c 的血红素配体的氧化还原状态。这种修饰会引起微妙的结构变化，表现为 N-Hcy-细胞色素 c 对胰蛋白酶、胰凝乳蛋白酶和链霉蛋白酶水解的抵抗力增强。然而，如圆二色光谱所示，没有观察到主要的二级结构扰动。我们的数据说明了 N-同型半胱氨酸化如何干扰含血红素蛋白质的功能。
Homocysteine (Hcy)-thiolactone mediates a post-translational incorporation of Hcy into protein in humans. Protein N-homocysteinylation is detrimental to protein structure and function and is linked to pathophysiology of hyperhomocysteinemia observed in humans and experimental animals. The modification by Hcy-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors. Previous work has shown that cytochrome c is very sensitive to Hcy-thiolactone, which causes formation of N-Hcy-cytochrome c multimers. However, it was unclear what sites in cytochrome c were prone to Hcy attachment and whether N-linked Hcy can affect the structure and redox function of cytochrome c. Here we show that 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) of cytochrome c are susceptible to N-homocysteinylation. We also show that N-homocysteinylation of 1 mol of lysine/mol of protein affects the redox state of the heme ligand of cytochrome c by rendering it reduced. The modification causes subtle structural changes, manifested as increased resistance of the N-Hcy-cytochrome c to proteolysis by trypsin, chymotrypsin, and Pronase. However, no major secondary structure perturbations were observed as shown by circular dichroism spectroscopy. Our data illustrate how N-homocysteinylation can interfere with the function of heme-containing proteins.