Barker Chérise D, Reda Torsten, Hirst Judy
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK.
Biochemistry. 2007 Mar 20;46(11):3454-64. doi: 10.1021/bi061988y. Epub 2007 Feb 27.
Complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria contains 45 different subunits and nine redox cofactors. NADH is oxidized by a noncovalently bound flavin mononucleotide (FMN), then seven iron-sulfur clusters transfer the two electrons to quinone, and four protons are pumped across the inner mitochondrial membrane. Here, we use protein film voltammetry to investigate the mechanisms of NADH oxidation and NAD+ reduction in the simplest catalytically active subcomplex of complex I, the flavoprotein (Fp) subcomplex. The Fp subcomplex was prepared using chromatography and contained the 51 and 24 kDa subunits, the FMN, one [4Fe-4S] cluster, and one [2Fe-2S] cluster. The reduction potential of the FMN in the enzyme's active site is lower than that of free FMN (thus, the oxidized state of the FMN is most strongly bound) and close to the reduction potential of NAD+. Consequently, the catalytic transformation is reversible. Electrocatalytic NADH oxidation by subcomplex Fp can be explained by a model comprising substrate mass transport, the Michaelis-Menten equation, and interfacial electron transfer kinetics. The difference between the "catalytic" potential and the FMN potential suggests that the flavin is reoxidized before NAD+ is released or that intramolecular electron transfer from the flavin to the [4Fe-4S] cluster influences the catalytic rate. NAD+ reduction displays a marked activity maximum, below which the catalytic rate decreases sharply as the driving force increases. Two possible models reproduce the observed catalytic waveshapes: one describing an effect from reducing the proximal [2Fe-2S] cluster and the other the enhanced catalytic ability of the semiflavin state.
来自牛心线粒体的复合物I（NADH：泛醌氧化还原酶）包含45种不同的亚基和9种氧化还原辅因子。NADH被非共价结合的黄素单核苷酸（FMN）氧化，然后七个铁硫簇将两个电子转移至醌，同时四个质子被泵过线粒体内膜。在此，我们使用蛋白质膜伏安法来研究复合物I最简单的具有催化活性的亚复合物即黄素蛋白（Fp）亚复合物中NADH氧化和NAD⁺还原的机制。Fp亚复合物通过色谱法制备，包含51 kDa和24 kDa的亚基、FMN、一个[4Fe - 4S]簇和一个[2Fe - 2S]簇。酶活性位点中FMN的还原电位低于游离FMN的还原电位（因此，FMN的氧化态结合最为紧密）且接近NAD⁺的还原电位。因此，催化转化是可逆的。亚复合物Fp对NADH的电催化氧化可以用一个包含底物质量传输、米氏方程和界面电子转移动力学的模型来解释。“催化”电位与FMN电位之间的差异表明，黄素在NAD⁺释放之前就被再氧化，或者从黄素到[4Fe - 4S]簇的分子内电子转移影响催化速率。NAD⁺还原表现出明显的活性最大值，在此值之下，随着驱动力增加，催化速率急剧下降。有两种可能的模型可以重现观察到的催化波形：一种描述了还原近端[2Fe - 2S]簇的影响，另一种描述了半黄素状态增强的催化能力。
