Biterova Ekaterina I, Turanov Anton A, Gladyshev Vadim N, Barycki Joseph J
Department of Biochemistry, University of Nebraska, Lincoln, NE 68588-0664, USA.
Proc Natl Acad Sci U S A. 2005 Oct 18;102(42):15018-23. doi: 10.1073/pnas.0504218102. Epub 2005 Oct 10.
Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine-containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a mitochondrial TrxR, TrxR2 (also known as TR3 and TxnRd2). Mouse TrxR2, in which the essential selenocysteine residue had been replaced with cysteine, was isolated as a FAD-containing holoenzyme and crystallized (2.6 A; R = 22.2%; R(free) = 27.6%). The addition of NADPH to the TrxR2 crystals resulted in a color change, indicating reduction of the active-site disulfide and formation of a species presumed to be the flavin-thiolate charge transfer complex. Examination of the NADP(H)-bound model (3.0 A; R = 24.1%; R(free) = 31.2%) indicates that an active-site tyrosine residue must rotate from its initial position to stack against the nicotinamide ring of NADPH, which is juxtaposed to the isoalloxazine ring of FAD to facilitate hydride transfer. Detailed analysis of the structural data in conjunction with a model of the unusual C-terminal selenenylsulfide suggests molecular details of the reaction mechanism and highlights evolutionary adaptations among reductases.
硫氧还蛋白还原酶（TrxR）是有效维持细胞氧化还原稳态所必需的一种酶，在对活性氧敏感的癌细胞中尤为重要。在哺乳动物中，不同的同工酶在细胞质和线粒体中发挥作用。通过一种复杂的机制，这些酶将还原当量从NADPH转移到结合的FAD，随后转移到活性位点二硫键。在哺乳动物TrxR中，二硫醇然后还原二聚体相对亚基含硒半胱氨酸的可移动C末端四肽。一旦被激活，C末端氧化还原中心还原硫氧还蛋白内的二硫键。在本报告中，我们展示了线粒体TrxR即TrxR2（也称为TR3和TxnRd2）的结构数据。将必需的硒半胱氨酸残基替换为半胱氨酸的小鼠TrxR2作为含FAD的全酶被分离并结晶（2.6埃；R = 22.2%；R（自由） = 27.6%）。向TrxR2晶体中添加NADPH导致颜色变化，表明活性位点二硫键被还原并形成一种推测为黄素 - 硫醇盐电荷转移复合物的物质。对结合NADP（H）的模型（3.0埃；R = 24.1%；R（自由） = 31.2%）的研究表明，一个活性位点酪氨酸残基必须从其初始位置旋转以与NADPH的烟酰胺环堆叠，该环与FAD的异咯嗪环并列以促进氢化物转移。结合不寻常的C末端硒代亚磺酸盐模型对结构数据的详细分析揭示了反应机制的分子细节，并突出了还原酶之间的进化适应性。
