The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria
The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria
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DOI：
10.1073/pnas.0510977103
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发表时间：
2006-05-16
影响因子：
11.1
通讯作者：
Hirst, Judy
中科院分区：
文献类型：
--
作者：
Kussmaul, Lothar;Hirst, Judy
NADH：泛醌氧化还原酶(复合体1)是线粒体中活性氧的主要来源，也是细胞氧化应激的重要贡献者。在这里，我们描述了从牛心线粒体中分离出来的络合物I产生超氧化物的动力学和分子机制，并确认它主要产生超氧化物，而不是过氧化氢。氧化还原滴定和电子顺磁共振谱排除了铁-硫团簇和黄素自由基作为超氧化物的来源，并且在没有质子动力的情况下，在周转过程中不会促进超氧化物的形成。因此，超氧化物是由一个电子从完全还原的黄素转移到O-2而形成的。由此产生的黄素自由基是不稳定的，所以剩余的电子可能被重新分配到铁-硫中心。超氧化物的生成速率是由O-2和空活性中心中的还原黄素之间的双分子反应决定的。因此，能够进行反应的黄素的比例是由前平衡点决定的，该平衡由NADH和NaDI的离解常数以及黄素和NaDI的还原电位决定。因此，NADH和NaDI的比例和浓度决定了超氧化物的形成速度。这一结果清楚地将我们对分离酶的机制与对完整线粒体的研究联系在一起，在完整线粒体中，当NaDI池减少时，超氧化物歧化酶的产生会增加。因此，我们的机制为建立复杂I缺陷与病理效应之间的因果联系奠定了基础。
NADH:ubiquinone oxicloreductase (complex 1) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O-2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O-2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preecluilibrium, determined by the dissociation constants of NADH and NADI, and the reduction potentials of the flavin and NADI. Consequently, the ratio and concentrations of NADH and NADI determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NADI pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects.