Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo 05508-000, Brazil.
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo 05508-090, Brazil.
J Biol Chem. 2019 Sep 20;294(38):14055-14067. doi: 10.1074/jbc.RA119.008825. Epub 2019 Jul 30.
2-Cys peroxiredoxins (Prxs) rapidly reduce HO, thereby acting as antioxidants and also as sensors and transmitters of HO signals in cells. Interestingly, eukaryotic 2-Cys Prxs lose their peroxidase activity at high HO levels. Under these conditions, HO oxidizes the sulfenic acid derivative of the Prx peroxidatic Cys (CSOH) to the sulfinate (CSO) and sulfonated (CSO) forms, redirecting the CSOH intermediate from the catalytic cycle to the hyperoxidation/inactivation pathway. The susceptibility of 2-Cys Prxs to hyperoxidation varies greatly and depends on structural features that affect the lifetime of the CSOH intermediate. Among the human Prxs, Prx1 has an intermediate susceptibility to HO and was selected here to investigate the effect of a physiological concentration of HCO/CO (25 mm) on its hyperoxidation. Immunoblotting and kinetic and MS/MS experiments revealed that HCO/CO increases Prx1 hyperoxidation and inactivation both in the presence of excess HO and during enzymatic (NADPH/thioredoxin reductase/thioredoxin) and chemical (DTT) turnover. We hypothesized that the stimulating effect of HCO/CO was due to HCO, a peroxide present in equilibrated solutions of HO and HCO/CO Indeed, additional experiments and calculations uncovered that HCO oxidizes CSOH to CSO with a second-order rate constant 2 orders of magnitude higher than that of HO ((1.5 ± 0.1) × 10 and (2.9 ± 0.2) × 10 m·s, respectively) and that HCO is 250 times more efficient than HO at inactivating 1% Prx1 per turnover. The fact that the biologically ubiquitous HCO/CO pair stimulates Prx1 hyperoxidation and inactivation bears relevance to Prx1 functions beyond its antioxidant activity.
2- 半胱氨酸过氧化物酶（Prx）迅速还原 HO，从而在细胞中充当抗氧化剂以及 HO 信号的传感器和转导器。有趣的是，真核生物 2- 半胱氨酸 Prx 在高 HO 水平下失去其过氧化物酶活性。在这些条件下，HO 将 Prx 过氧催化半胱氨酸（CSOH）的亚磺酸衍生物氧化为亚磺酸盐（CSO）和磺酸盐（CSO ）形式，将 CSOH 中间体从催化循环重定向到过氧氧化/失活途径。2- 半胱氨酸 Prx 对过氧氧化的敏感性差异很大，这取决于影响 CSOH 中间体寿命的结构特征。在人类 Prx 中，Prx1 对 HO 的敏感性中等，因此选择在这里研究生理浓度的 HCO/CO（25 mM）对其过氧氧化的影响。免疫印迹、动力学和 MS/MS 实验表明，HCO/CO 增加了 Prx1 在 HO 过量存在下以及在酶促（NADPH/硫氧还蛋白还原酶/硫氧还蛋白）和化学（DTT）循环过程中的过氧氧化和失活。我们假设 HCO/CO 的刺激作用是由于 HCO 引起的，HCO 是 HO 和 HCO/CO 平衡溶液中的过氧化物。事实上，额外的实验和计算揭示了 HCO 将 CSOH 氧化为 CSO 的二级速率常数比 HO 高 2 个数量级（分别为（1.5 ± 0.1）×10 和（2.9 ± 0.2）×10 m·s），并且 HCO 在每轮循环中失活 1%Prx1 的效率比 HO 高 250 倍。生物普遍存在的 HCO/CO 对 Prx1 过氧氧化和失活的刺激作用与 Prx1 的抗氧化活性以外的功能有关。
