Samhan-Arias Alejandro K, Gutierrez-Merino Carlos
Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, 06006 Badajoz, Spain.
Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, 06006 Badajoz, Spain.
Free Radic Biol Med. 2014 Aug;73:174-89. doi: 10.1016/j.freeradbiomed.2014.04.033. Epub 2014 May 6.
Cytochrome b5 reductase (Cb5R) is a pleiotropic flavoprotein that catalyzes multiple one-electron reduction reactions with various redox partners in cells. In earlier work from our laboratory, we have shown its implication in the generation of reactive oxygen species (ROS), primarily a superoxide anion overshoot peak, which plays a major role as a triggering event for the acceleration of apoptosis in cerebellar granule neurons in culture. However, the results obtained in that work did not allow us to exclude the possibility that this superoxide anion production could be derived from Cb5R acting in concert with other cellular components. In this work, we have purified Cb5R from pig liver and we have experimentally shown that this enzyme catalyzed NADH-dependent production of superoxide anion, assayed with cytochrome c and nitroblue tetrazolium as detection reagents for this particular ROS. The basic kinetic parameters for this novel NADH-dependent activity of Cb5R at 37°C are Vmax = 3.0 ± 0.5 μmol/min/mg of purified Cb5R and KM(NADH) = 2.8 ± 0.3 μM NADH. In addition, we report that apocynin, a widely used inhibitor of nonmitochondrial ROS production in mammalian cell cultures and tissues, is a potent inhibitor of purified Cb5R activity at the concentrations used in the experiments done with cell cultures. In the presence of apocynin the KM(NADH) value of Cb5R increases, and docking simulations indicate that apocynin can bind to a site near to or partially overlapping the NADH binding site of Cb5R. Other ROS, such as nitric oxide and peroxynitrite, have inhibitory effects on purified Cb5R, providing the basis for a feedback cellular protection mechanism through modulation of excessive extramitochondrial superoxide anion production by Cb5R. Both kinetic assays and docking simulations suggest that nitric oxide-induced nitrosylation (including covalent adduction of nitroso functional groups) of Cb5R cysteines and peroxynitrite-induced tyrosine nitration and cysteine oxidation modified the conformation of the NADH binding domain leading to a decreased affinity of Cb5R for NADH.
细胞色素b5还原酶（Cb5R）是一种多效性黄素蛋白，它在细胞内与各种氧化还原伙伴催化多个单电子还原反应。在我们实验室早期的工作中，我们已经表明它与活性氧（ROS）的产生有关，主要是超氧阴离子过冲峰，这在体外培养的小脑颗粒神经元凋亡加速的触发事件中起主要作用。然而，该工作中获得的结果并不能让我们排除这种超氧阴离子产生可能源自Cb5R与其他细胞成分协同作用的可能性。在这项工作中，我们从猪肝中纯化了Cb5R，并通过实验表明该酶催化依赖NADH的超氧阴离子产生，使用细胞色素c和硝基蓝四唑作为该特定ROS的检测试剂进行测定。Cb5R在37°C时这种新的依赖NADH活性的基本动力学参数为：Vmax = 3.0 ± 0.5 μmol/分钟/毫克纯化的Cb5R，KM（NADH） = 2.8 ± 0.3 μM NADH。此外，我们报告说，阿朴吗啡，一种在哺乳动物细胞培养物和组织中广泛使用的非线粒体ROS产生抑制剂，在细胞培养实验中使用的浓度下是纯化的Cb5R活性的有效抑制剂。在阿朴吗啡存在下，Cb5R的KM（NADH）值增加，对接模拟表明阿朴吗啡可以结合到靠近Cb5R的NADH结合位点或与之部分重叠的位点。其他ROS，如一氧化氮和过氧亚硝酸盐，对纯化的Cb5R有抑制作用，为通过调节Cb5R过量的线粒体外超氧阴离子产生的反馈细胞保护机制提供了基础。动力学测定和对接模拟均表明，一氧化氮诱导的Cb5R半胱氨酸亚硝化（包括亚硝基官能团的共价加成）以及过氧亚硝酸盐诱导的酪氨酸硝化和半胱氨酸氧化改变了NADH结合域的构象，导致Cb5R对NADH的亲和力降低。
