Mechanism of tyrosine 69 in the flavin domain modulating electron transfer efficiency in BVU5 azoreductase

Abstract

The azoreductase BVU5 is a flavoprotein dependent on NAD(P)H/FMN-mediated electron transfer; However, the functional mechanism of key residues within its flavin domain remains unclear. This study identified the conserved residue Tyr⁶⁹ through bioinformatics analysis and constructed Y69F and Y69C mutants. Enzymatic assays demonstrated that mutants exhibited significantly lower decolorization rates than the BVU5 enzyme across 12 dye molecules, including azo, anthraquinone, and triphenylmethane dyes. For example, when decolorizing Reactive Black 5 (RB5) for 2 h: Y69F mutant achieved 60 %-65 % decolorization, Y69C mutant reached 55 %-60 % decolorization, both markedly lower than the 80 %-85 % efficiency of BVU5.The decolorization hierarchy remained azo dyes > triphenylmethane > anthraquinone dyes. Molecular docking revealed that mutations reconfigured FMN-binding patterns. Although Y69F enlarged the substrate-binding pocket, it failed to enhance the degradation efficiency of the bulky dye Chlorazol Black E. This critical contradiction indicates that substrate binding is not the limiting factor. Combined with evidence such as the lighter color of the mutant enzyme solutions, increased A280/A450 ratios, and enzyme dosage experiments, this study confirms that Tyr⁶⁹ is a key residue that sustains electron transfer efficiency by maintaining the FMN-binding conformation, thereby determining the decolorization performance. Consequently, electron transfer efficiency, rather than substrate binding, is the primary mechanism influencing the catalytic function of BVU5.