Thiol peroxidases (TP) are ubiquitous and abundant antioxidant proteins of the peroxiredoxin and glutathione peroxidase families that can catalytically and rapidly reduce biologically relevant peroxides, such as hydrogen peroxide and peroxynitrite. However, the TP catalytic cycle is complex, depending on multiple redox reactions and partners, and is subjected to branching and competition points that may limit their peroxide reductase activity in vivo. The goals of the present study were to demonstrate peroxynitrite reductase activity of TP members in live cells in real time and to evaluate its catalytic characteristics. To these ends, we developed a simple fluorescence assay using coumarin boronic acid (CBA), exploiting that fact that TP and CBA compete for peroxynitrite, with the expectation that higher TP peroxynitrite reductase activity will lower the CBA oxidation. TP peroxynitrite reductase activity was evaluated by comparing CBA oxidation in live wild type and genetically modified Δ8 (TP-deficient strain) and Δ8+TSA1 (Δ8 strain that expresses only one TP member, the TSA1 gene) Saccharomyces cerevisiae strains. The results showed that CBA oxidation decreased with cell density and increased with increasing peroxynitrite availability. Additionally, the rate of CBA oxidation decreased in the order Δ8 > Δ8+TSA1 > WT strains both in control and glycerol-adapted (expressing higher TP levels) cells, showing that the CBA competition assay could reliably detect peroxynitrite in real time in live cells, comparing CBA oxidation in strains with reduced and increased TP expression. Finally, there were no signs of compromised TP peroxynitrite reductase activity during experimental runs, even at the highest peroxynitrite levels tested. Altogether, the results show that TP is a major component in the defense of yeast against peroxynitrite insults under basal and increasing stressful conditions.

中文翻译：

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硫醇过氧化物酶是酿酒酵母细胞中亚硝酸盐含量的主要调节剂。

硫醇过氧化物酶（TP）是过氧化物酶和谷胱甘肽过氧化物酶家族中普遍存在且丰富的抗氧化剂蛋白，可以催化并快速还原生物学上相关的过氧化物，例如过氧化氢和过氧亚硝酸盐。然而，TP催化循环是复杂的，取决于多个氧化还原反应和伙伴，并且经受分支和竞争点，这可能限制它们在体内的过氧化物还原酶活性。本研究的目的是实时证明活细胞中TP成员的过氧亚硝酸盐还原酶活性并评估其催化特性。为此，我们利用香豆素硼酸（CBA）开发了一种简单的荧光测定法，利用了TP和CBA竞争过氧亚硝酸盐这一事实，期望更高的TP过氧亚硝酸盐还原酶活性将降低CBA氧化。通过比较活野生型和基因修饰的Δ8（TP缺陷菌株）和Δ8+ TSA1（仅表达一个TP成员，TSA1基因的Δ8菌株）酿酒酵母菌株中的CBA氧化来评估TP过亚硝酸盐还原酶活性。结果表明，CBA氧化随着细胞密度的增加而降低，并随着过氧亚硝酸盐可用性的增加而增加。此外，在对照和甘油适应（表达更高的TP水平）细胞中，CBA氧化速率均按Δ8>Δ8+ TSA1> WT菌株的顺序降低，表明CBA竞争测定法能够可靠地实时检测活细胞中的亚硝酸盐，比较了TP表达减少和增加的菌株中的CBA氧化。最后，即使在测试的最高过氧亚硝酸盐水平下，在实验过程中也没有损害TP过氧亚硝酸盐还原酶活性的迹象。总而言之，结果表明在基础和不断增加的压力条件下，TP是酵母防御过氧亚硝酸盐侵害的主要成分。