pH-Induced Alteration and Oxidative Destruction of Heme in Purified Chromaffin Granule Cytochrome b561: Implications for the Oxidative Stress in Catecholaminergic Neurons

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• 作者：Srimevan Wanduragala ; D. Shyamali Wimalasena ; Donovan C. Haines ; Pawan K. Kahol ; and Kandatege Wimalasena
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：April 1, 2003
• 年：2003
• 卷：42
• 期：12
• 页码：3617 - 3626
• 全文大小：169K
• 年卷期：v.42,no.12(April 1, 2003)
• ISSN：1520-4995

文摘

The transmembrane hemoprotein, cytochrome b₅₆₁ (b₅₆₁), in the neuroendocrine secretoryvesicles is shown to shuttle electrons from the cytosolic ascorbate (Asc) to the intravesicular matrix toprovide reducing equivalents for the dopamine -monooxygenase (DM) reaction. Intravesicular Ascmay also play a role in relieving catecholamine-induced oxidative stress in catecholaminergic neurons. Inthe present study, we have examined the alteration of purified oxidized b₅₆₁ (b_561,ox) under mild alkalineconditions to probe the structural and functional characteristics of the protein, using UV-vis and EPRspectroscopic and kinetic techniques. Our results show that low spin heme in oxidized b₅₆₁ (b_561,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,A_t = A_o(1 - e^-kt) and A_t = A_oe^-kt, respectively. More than half of the Asc nonreducible altered b₅₆₁ couldbe converted back to the native b₅₆₁ by pH adjustment followed by dithionite reduction, suggesting thereversibility of the process. The heme center of the transformed Asc nonreducible protein is completelybleached instantaneously by dithionite in the presence of atmospheric oxygen, which appears to be mediatedby molecular oxygen and/or hydrogen peroxide. These results demonstrate that the heme centers of theprotein are susceptible to the pH-induced alteration and oxidative destruction, raising some questionsregarding 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 destructionof heme under oxidative conditions may play a significant role in the amplification of oxidative stress incatecholaminergic neurons.