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Abstract

Upon oxygen activation, the non-heme diiron enzymes can generate various active species for oxidative transformations. In this work, the catalytic mechanism of the diiron active site (heme-oxygenase-like diiron oxidase (HDO) domain) in SznF has been comprehensively studied by molecular docking, classical molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) MD simulations, and hybrid QM/MM calculations. The HDO domain of SznF catalyzes the selective hydroxylation of N^ω-methyl-l-arginine (l-NMA) to generate N^δ-hydroxy-N^ω-methyl-l-Arg (l-HMA) and N^δ,N^ω-dihydroxy-N^ω,-methyl-l-Arg (l-DHMA), which is a key step in the synthesis of the nitrosourea pharmacophore of the pancreatic cancer drug streptozotocin (SZN). Our study shows that the peroxo-diiron(III/III) intermediate in Sznf maintains a butterfly-like conformation, while the further protonation of the diiron(III/III) intermediate is found to be inaccessible and unfavorable thermodynamically. Among various mechanisms, we found that the most favorable mechanism involves the nucleophilic attack of the guanidium group onto the peroxo group of P1, which drives the heterolytic cleavage of the O–O bond. Moreover, the selectivity of N-hydroxylation by the peroxo-diiron(III/III) intermediate can be fully supported by MD simulations, suggesting that the peroxo-diiron(III/III) is the reactive intermediate for N-hydroxylation in SznF. The present study expands our understanding on the O₂ activation and N-hydroxylation by the non-heme diiron enzymes.