Selenocysteine (Sec) is a crucial component of mammalian thioredoxin reductase (TrxR) where it serves as a nucleophile for disulfide bond rupture in thioredoxin (Trx). Generation of the reduced state of Sec in TrxR requires consecutive two electron transfer steps, namely: (i) from NADPH to flavin adenine dinucleotide, (ii) from reduced flavin to the disulfide bond Cys⁵⁹‐S‐S‐Cys⁶⁴, and finally (iii) from Cys⁵⁹ and Cys⁶⁴ to the selenosulfide bond Cys⁴⁹⁷‐S‐Se‐Sec⁴⁹⁸. In this work, we studied the reaction between reduced riboflavin (RibH2) and selenocystine (Sec‐Sec), an oxidized form of Sec. The interaction between RibH2 and Sec‐Sec proceeded relatively slowly in comparison with its reverse reaction, that is, reduction of riboflavin (Rib) by Sec. The rate constant for the reaction between RibH2 and Sec‐Sec was (7.9 ± 0.1) × 10⁻² M⁻¹ s⁻¹ (pH 7.0, 25.0°C). The reaction between Rib and Sec proceeded via two steps, namely, a rapid reversible binding of Rib to Sec having a protonated selenol group to form a Sec‐Rib complex, followed by nucleophilic attack of Sec‐Rib by a second Sec molecule harboring a deprotonated selenol group. The equilibrium constant for the overall reduction process of Rib by Sec is (1.2 ± 0.1) × 10⁶ M⁻¹ (25.0°C). The finding that the interaction of RibH2 with oxidized selenol is reversible with its equilibrium favored toward the reverse reaction provides an additional explanation for the exceptional mechanism of the mammalian Trx/TrxR system involving transient reduction of a disulfide bond.

中文翻译：

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还原核黄素与硒代半胱氨酸反应的研究

硒代半胱氨酸（Sec）是哺乳动物硫氧还蛋白还原酶（TrxR）的重要组成部分，它充当硫氧还蛋白（Trx）中二硫键断裂的亲核试剂。在TrxR中生成Sec还原态需要连续两个电子转移步骤，即：（i）从NADPH到黄素腺嘌呤二核苷酸，（ii）从黄素还原到二硫键Cys ⁵⁹ -S-S-Cys ⁶⁴，最后（iii）从Cys ⁵⁹和Cys ⁶⁴到硒代硫键Cys ⁴⁹⁷ -S-Se-Sec ⁴⁹⁸。在这项工作中，我们研究了还原型核黄素（RibH2）与硒代半胱氨酸（Sec-Sec）之间的反应，Sec-Sec是氧化形式的Sec。与RibH2和Sec-Sec之间的相互作用相比，它的逆反应（即Sec减少核黄素（Rib））的反应进行得相对缓慢。RibH2与Sec-Sec之间反应的速率常数为（7.9±0.1）×10 ^-2  M ^-1 s ^-1（pH 7.0，25.0°C）。Rib和Sec之间的反应通过两个步骤进行，即Rib与具有质子化硒醇基的Sec的快速可逆结合以形成Sec-Rib络合物，然后通过第二个带有去质子化的Sec分子对Sec-Rib进行亲核攻击硒醇组。Sec的Rib整个还原过程的平衡常数为（1.2±0.1）×10⁶  M ^-1（25.0°C）。RibH2与氧化硒醇的相互作用是可逆的，其平衡有利于逆反应，这一发现为哺乳动物Trx / TrxR系统涉及二硫键瞬时还原的异常机理提供了额外的解释。