Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation
Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation
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DOI：
10.1074/jbc.m503936200
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发表时间：
2005-11-11
影响因子：
4.8
通讯作者：
Zweier, JL
中科院分区：
文献类型：
--
作者：
Chen, YR;Chen, CL;Zweier, JL
线粒体超氧化物（O-2（自由基阴离子）。）产生是氧化性细胞损伤的重要介质。虽然NADH脱氢酶（NDH）是这种O-2（自由基阴离子）产生的关键位点;但其O-2（自由基阴离子）产生的机制尚不清楚。因此，NDH在介导的O-2（自由基阴离子）产生的催化功能进行了研究EPR自旋捕获。在NADH存在下，观察到NDH产生O-2（自由基阴离子），并被氯化二苯基碘铵（DPI）抑制，表明参与NDH的FMN结合位点。FMN的加入增加了O-2（自由基阴离子）的产生。破坏铁硫簇的半胱氨酸配体减少O-2（自由基阴离子）的产生，这表明该网站的次要作用。这种抑制作用被FMN逆转。然而，FMN除了不能逆转的抑制NDH的DPI或热变性，表明参与FMN和它的FMN结合蛋白部分的催化O-2（自由基阴离子）的产生。O-2（自由基阴离子）生产NDH也诱导自我失活。用抗DMPO抗体和随后的质谱进行免疫自旋捕获，以确定NDH的氧化损伤的网站。在51 kDa亚基上检测到DMPO加合物，并且是O-2（自由基阴离子）依赖性的。烷基化的半胱氨酸残基的NDH显着抑制NDH-DMPO自旋加合物的形成，表明参与蛋白质巯基自由基。51-kDa多肽的胰蛋白酶消化物的LC/MS/MS分析显示，半胱氨酸（Cys（206））和酪氨酸（Tyr（177））是NDH衍生的蛋白质自由基形成的特异性位点。因此，51-kDa亚基的两个结构域Gly（200-）Ala-Gly-Ala-Tyr-Ile-Cys（206）- Gly-Glu-Glu- Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys（219）和Ala（176）-Tyr（177）- Glu-Ala-Gly-Leu-Ile-Gly-Lys（184）被证明对氧化攻击敏感，并且它们的氧化修饰导致电子转移活性降低。
Mitochondrial superoxide (O-2(radical anion).) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O-2(radical anion) production; its mechanism of O-2(radical anion) generation is not known. Therefore, the catalytic function of NDH in the mediation of O-2(radical anion) generation was investigated by EPR spin-trapping. In the presence of NADH, O-2(radical anion) generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased O-2(radical anion) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O-2(radical anion) generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of O-2(radical anion) generation. O-2(radical anion) production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was O-2(radical anion)-dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys(206)) and tyrosine (Tyr(177)) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly(200-) Ala-Gly-Ala-Tyr-Ile-Cys(206)- Gly-Glu-Glu- Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys(219) and Ala(176)-Tyr(177)- Glu-Ala-Gly-Leu-Ile-Gly-Lys(184), were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.