Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
Centro de Investigaciones Biomédicas (CEINBIO), Universidad de la República, Montevideo, Uruguay.
FEBS J. 2024 Feb;291(4):778-794. doi: 10.1111/febs.17006. Epub 2023 Dec 7.
We have studied the reduction reactions of two cytosolic human peroxiredoxins (Prx) in their disulfide form by three thioredoxins (Trx; two human and one bacterial), with the aim of better understanding the rate and mechanism of those reactions, and their relevance in the context of the catalytic cycle of Prx. We have developed a new methodology based on stopped-flow and intrinsic fluorescence to study the bimolecular reactions, and found rate constants in the range of 10 -10 m s in all cases, showing that there is no marked kinetic preference for the expected Trx partner. By combining experimental findings and molecular dynamics studies, we found that the reactivity of the nucleophilic cysteine (C ) in the Trx is greatly affected by the formation of the Prx-Trx complex. The protein-protein interaction forces the C thiolate into an unfavorable hydrophobic microenvironment that reduces its hydration and results in a remarkable acceleration of the thiol-disulfide exchange reactions by more than three orders of magnitude and also produces a measurable shift in the pK of the C . This mechanism of activation of the thiol disulfide exchange may help understand the reduction of Prx by alternative reductants involved in redox signaling.
我们研究了两种胞质人过氧化物酶（Prx）在其二硫键形式下被三种硫氧还蛋白（Trx；两种人源和一种细菌源）还原的反应，目的是更好地理解这些反应的速率和机制，以及它们在 Prx 催化循环背景下的相关性。我们开发了一种基于停流和本征荧光的新方法来研究双分子反应，在所有情况下都发现了 10 -10 m s 的速率常数，表明对于预期的 Trx 伴侣没有明显的动力学偏好。通过结合实验结果和分子动力学研究，我们发现 Trx 中亲核半胱氨酸（C ）的反应性受到 Prx-Trx 复合物形成的极大影响。蛋白质-蛋白质相互作用迫使 C 硫醇盐进入不利的疏水环境，从而降低其水合作用，并导致硫醇-二硫键交换反应显著加速超过三个数量级，并且还会导致 C 的 pK 值发生可测量的变化。这种硫醇-二硫键交换的激活机制可能有助于理解参与氧化还原信号的替代还原剂对 Prx 的还原。
