pH-induced alteration and oxidative destruction of heme in purified chromaffin granule cytochrome b(561): implications for the oxidative stress in catecholaminergic neurons.
pH-induced alteration and oxidative destruction of heme in purified chromaffin granule cytochrome b(561): implications for the oxidative stress in catecholaminergic neurons.
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pH 诱导的纯化嗜铬颗粒细胞色素 b(561) 中血红素的改变和氧化破坏：对儿茶酚胺能神经元氧化应激的影响。
DOI：
10.1021/bi0206661
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Wimalasena,Kandatege
中科院分区：
文献类型：
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作者：
Wanduragala,Srimevan;Wimalasena,DShyamali;Haines,DonovanC;Kahol,PawanK;Wimalasena,Kandatege
神经内分泌分泌囊泡中的跨膜血红蛋白细胞色素b561(B561)可以将电子从胞浆抗坏血酸(AsC)传递到囊泡内基质，为多巴胺β-单加氧酶(DβM)反应提供还原等价物。泡内ASC也可能在减轻儿茶酚胺能神经元的氧化应激中发挥作用。在本研究中，我们研究了纯化的氧化B561(B561，OX)在弱碱性条件下的变化，并利用UV−Vis、EPR光谱和动力学技术探讨了该蛋白的结构和功能特征。我们的结果表明，氧化的b561(b561，ox)中的低自旋血红素很容易转变为改变的高自旋形式，然后缓慢地转变为与pH、温度和时间有关的Asc不可还原形式，这可以分别用单指数速率方程At=Ao(1−e-kt)和At=AOe-kt来描述。ASC不可还原蚀变b561的一半以上可通过调节pH和二亚硫酸盐还原还原成天然b561，这表明该过程是可逆的。转化的ASC不可还原蛋白的血红素中心在大气氧气存在下被二亚硫酸盐瞬间完全漂白，这似乎是由分子氧和/或过氧化氢介导的。这些结果表明，蛋白质的血红素中心对pH诱导的改变和氧化破坏很敏感，这对提出的b561的一个碱性不稳定，两个血红素模型提出了一些问题[Tsubaki，M.；Nakayama，M.；Okuyama，E.；Ichikawa，Y.(1997)J.Biol。化学成分272,23206−23210]。PH诱导的氧化条件下血红素的改变和破坏可能在儿茶酚胺能神经元氧化应激的放大中起重要作用。
The transmembrane hemoprotein, cytochrome b561(b561), in the neuroendocrine secretory vesicles is shown to shuttle electrons from the cytosolic ascorbate (Asc) to the intravesicular matrix to provide reducing equivalents for the dopamine β-monooxygenase (DβM) reaction. Intravesicular Asc may also play a role in relieving catecholamine-induced oxidative stress in catecholaminergic neurons. In the present study, we have examined the alteration of purified oxidized b561(b561,ox) under mild alkaline conditions to probe the structural and functional characteristics of the protein, using UV−vis and EPR spectroscopic and kinetic techniques. Our results show that low spin heme in oxidized b561(b561,ox) readily transforms to an altered high spin form and then slowly to an Asc nonreducible form, in a pH-, temperature-, and time-dependent manner, which can be described by single-exponential rate equations,At=Ao(1 − e-kt) andAt=Aoe-kt, respectively. More than half of the Asc nonreducible altered b561could be converted back to the native b561by pH adjustment followed by dithionite reduction, suggesting the reversibility of the process. The heme center of the transformed Asc nonreducible protein is completely bleached instantaneously by dithionite in the presence of atmospheric oxygen, which appears to be mediated by molecular oxygen and/or hydrogen peroxide. These results demonstrate that the heme centers of the protein are susceptible to the pH-induced alteration and oxidative destruction, raising some questions regarding the proposed one alkaline labile, two-heme model of b561[Tsubaki, M.; Nakayama, M.; Okuyama, E.; Ichikawa, Y. (1997)J. Biol. Chem.272,23206−23210]. The pH-induced alteration and the destruction of heme under oxidative conditions may play a significant role in the amplification of oxidative stress in catecholaminergic neurons.