Interaction between flavin mononucleotide-containing azoreductase and azo dyes

Azoreductase, a flavin mononucleotide-containing oxidoreductase from <i>Escherichia coli</i>, can catalyze the reduction of azo dyes to form aromatic amine compounds. Few spectroscopic studies have explored the binding mode of azo dyes or the role of the arginine at site 59 in Azoreductase. In this article, protein engineering strategy has been used to construct one mutant in which the arginine residue at site 59 was mutated to glycine. Fluorescence spectroscopic data showed that the addition of Methyl Red and Methyl Orange resulted in the fluorescence quenching of the cofactor flavin mononucleotide bound to Azoreductase. The association constant was fitted using the standard binding equation instead of the Stern-Volmer equation. The results showed that the mutation from the arginine to glycine at site 59 weakened the association constant from 2.21 × 10⁵ L.mol⁻¹ to 4.55 × 10⁴ L.mol⁻¹ at 25°C. A similar phenomenon was also observed when Methyl Orange was used as a substrate. In each case, the association constant tended to decrease as the temperature increased from 25°C to 37°C. Thermodynamic parameter analysis revealed that the interaction type changed from a van der Waals interaction (between Azoreductase and the dyes) to a hydrogen bonding interaction (between the mutant and the dyes). Moleculcar docking was also performed in this work to provide some support for the binding mode and binding stability between Azoreductase/mutant and azo dyes.

偶氮还原酶（Azoreductase）是一种来源于大肠杆菌（Escherichia coli）的含黄素单核苷酸氧化还原酶，可催化偶氮染料还原生成芳香胺类化合物。目前针对偶氮染料与该酶的结合模式，以及偶氮还原酶第59位精氨酸残基的作用所开展的光谱学研究尚少。本文采用蛋白质工程策略构建了一株突变体，将偶氮还原酶第59位的精氨酸残基突变为甘氨酸。荧光光谱数据显示，加入甲基红（Methyl Red）与甲基橙（Methyl Orange）后，偶氮还原酶结合的辅因子黄素单核苷酸会发生荧光猝灭。本研究采用标准结合方程而非斯特恩-沃尔默（Stern-Volmer）方程拟合结合常数。结果表明，在25℃条件下，第59位精氨酸突变为甘氨酸后，结合常数从2.21×10⁵ L·mol⁻¹降至4.55×10⁴ L·mol⁻¹。以甲基橙为底物时也观察到了类似现象。两种情况下，结合常数均随温度从25℃升至37℃而呈下降趋势。热力学参数分析显示，相互作用类型发生了变化：野生型偶氮还原酶与染料间为范德华相互作用（van der Waals interaction），而突变体与染料间则为氢键相互作用（hydrogen bonding interaction）。本研究还开展了分子对接（molecular docking）实验，为偶氮还原酶/突变体与偶氮染料的结合模式及结合稳定性提供了佐证。