2,4,6-trinitrotoluene (TNT) is a persistent pollutant; for removing it from environment, the biodegradation becomes an economical and environmentally friendly alternative. However, TNT is difficult to naturally mineralize due to the stability of benzene ring. Interestingly, the hydride metabolite (Hˉ-TNT) of TNT can produce dearomatized products by the biotransformation of old yellow enzyme (OYE) family, which is highly different with TNT. It is a promising strategy for the ring-opening degradation of TNT. Here, we explore the biotransformation difference of TNT and Hˉ-TNT by OYE family. The results show that the electron-withdrawing ability of nitro groups makes them obtain the majority of negative charges of Hˉ-TNT, which promotes the strong interaction between the substrate and key residues. It in turn promotes the formation of π-π stacking configuration between Hˉ-TNT and the flavin mononucleotide (FMN) cofactor, which is a precondition of the aromatic ring reduction. As a result, the aromatic ring reduction occupies an absolute advantage, while the nitro reduction is suppressed. The conclusions are in line with previous understanding and can explain experimental phenomena. This work can provide a valuable reference for the combined strategy of mineralizing TNT.

2,4,6-三硝基甲苯（TNT）是一种持久性污染物；为了将其从环境中去除，生物降解成为一种经济和环保的替代方法。然而，由于苯环的稳定性，TNT难以自然矿化。有趣的是，TNT的氢化物代谢物（Hˉ-TNT）可以通过旧的黄色酶（OYE）家族的生物转化产生脱芳烃化的产物，这与TNT有很大的不同。对于TNT的开环降解而言，这是一种有前途的策略。在这里，我们探讨了OYE家族对TNT和Hˉ-TNT的生物转化差异。结果表明，硝基的吸电子能力使它们获得H + -TNT的大部分负电荷，从而促进了底物与关键残基之间的强相互作用。反过来，它促进了H + -TNT与黄素单核苷酸（FMN）辅因子之间的π-π堆积构型是芳环还原的前提。结果，芳环还原具有绝对优势，而硝基还原被抑制。结论与先前的理解一致，并且可以解释实验现象。这项工作可以为TNT矿化的联合策略提供有价值的参考。