Chen Yeong-Renn, Chen Chwen-Lih, Zhang Liwen, Green-Church Kari B, Zweier Jay L
Department of Internal Medicine, Davis Heart & Lung Research Institute, Division of Cardiovascular Medicine, The Ohio State University, Columbus, 43210, USA.
J Biol Chem. 2005 Nov 11;280(45):37339-48. doi: 10.1074/jbc.M503936200. Epub 2005 Sep 8.
Mitochondrial superoxide (O(2)(.)) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O(2)(.) production; its mechanism of O(2)(.) generation is not known. Therefore, the catalytic function of NDH in the mediation of O(2)(.) generation was investigated by EPR spin-trapping. In the presence of NADH, O(2)(.) 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)(.) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O(2)(.) 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)(.) generation. O(2)(.) 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)(.)-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.
线粒体超氧阴离子（O₂⁻）的产生是细胞氧化损伤的重要介质。虽然NADH脱氢酶（NDH）是这种O₂⁻产生的关键位点，但其O₂⁻生成机制尚不清楚。因此，通过电子顺磁共振自旋捕获技术研究了NDH在介导O₂⁻生成中的催化功能。在NADH存在的情况下，观察到NDH产生O₂⁻，并且被二苯基氯化碘鎓（DPI）抑制，这表明NDH的FMN结合位点参与其中。添加FMN增加了O₂⁻的产生。铁硫簇的半胱氨酸配体的破坏减少了O₂⁻的生成，表明该位点起次要作用。添加FMN可逆转这种抑制作用。然而，添加FMN不能逆转DPI或热变性对NDH的抑制作用，这表明FMN及其FMN结合蛋白部分都参与了O₂⁻生成的催化过程。NDH产生O₂⁻也会导致自身失活。使用抗DMPO抗体进行免疫自旋捕获并随后进行质谱分析来确定NDH氧化损伤的位点。在51 kDa亚基上检测到DMPO加合物，且其依赖于O₂⁻。NDH半胱氨酸残基的烷基化显著抑制了NDH-DMPO自旋加合物的形成，表明蛋白质硫自由基参与其中。对51 kDa多肽的胰蛋白酶消化产物进行液相色谱/串联质谱分析表明，半胱氨酸（Cys₂₀₆）和酪氨酸（Tyr₁₇₇）是NDH衍生的蛋白质自由基形成的特定位点。因此，51 kDa亚基的两个结构域，即Gly₂₀₀-Ala-Gly-Ala-Tyr-Ile-Cys₂₀₆-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys₂₁₉和Ala₁₇₆-Tyr₁₇₇-Glu-Ala-Gly-Leu-Ile-Gly-Lys₁₈₄，被证明易受氧化攻击，并且它们的氧化修饰导致电子转移活性降低。
