Breaking the\nMyth of Enzymatic Azoreduction

Flavin-dependent azoreductases have been applied to a\nwide range\nof tasks from decolorizing numerous azo dyes to releasing azo-conjugated\nprodrugs. A general narrative reiterated in much of the literature\nsuggests that this enzyme promotes sequential reduction of both the\nazo-containing substrate and its corresponding hydrazo product to\nrelease the aryl amine components while consuming two equivalents\nof NAD(P)H. Indeed, such aryl amines can be formed by incubation of\ncertain azo compounds with azoreductases, but the nature of the substrates\ncapable of this apparent azo bond lysis remained unknown. We have\nnow prepared a set of azobenzene derivatives and characterized their\nturnover and products after treatment with azoreductase from Escherichia coli to discover the structural basis\nregulating aryl amine formation. Without resonance donation by aryl\nsubstituents, reduction ceases at the hydrazo product. This indicates\nthat azoreductases do not act on the hydrazo bond. Instead, aryl amine\nformation depends on a spontaneous hydrazo bond lysis that is promoted\nby resonance stabilization and subsequent reduction of the quinone-like\nintermediate by azoreductase. Experimental and computational approaches\nconfirm the substituent dependence of this process. With knowledge\nof this requirement, full release of aryl amines from azo-conjugates\ncan now be designed and applied with confidence.