Mechanistic Insights into the N-Hydroxylations Catalyzed by the Binuclear Iron Domain of SznF Enzyme: Key Piece in the Synthesis of Streptozotocin

SznF, a member of the emerging family of heme-oxygenase-like (HO-like) di-iron oxidases and oxygenases, employs two distinct domains to catalyze the conversion of N omega-methyl-L-arginine (L-NMA) into N-nitroso-containing product, which can subsequently be transformed into streptozotocin. Using unrestricted density functional theory (UDFT) with the hybrid functional B3LYP, we have mechanistically investigated the two sequential hydroxylations of L-NMA catalyzed by SznF's binuclear iron central domain. Mechanism B primarily involves the O-O bond dissociation, forming Fe(IV)=O, induced by the H+/e- introduction to the FeA side of mu-1,2-peroxo-Fe2(III/III), the substrate hydrogen abstraction by Fe(IV)=O, and the hydroxyl rebound to the substrate N radical. The stochastic addition of H+/e- to the FeB side (mechanism C) can transition to mechanism B, thereby preventing enzyme deactivation. Two other competing mechanisms, involving the direct O-O bond dissociation (mechanism A) and the addition of H2O as a co-substrate (mechanism D), have been ruled out. Enzyme catalysis: The consecutive hydroxylation mechanism of N omega-methyl-L-arginine (L-NMA) catalyzed by SznF's di-iron domain is presented. Initiated by the peroxo-Fe2(III/III) intermediate, the reaction involves proton/electron (H+/e-) addition on either side of this intermediate facilitating the generation of N delta-hydroxy-N omega-methyl-L-arginine (L-HMA) and N delta-hydroxy-N omega-hydroxy-N omega-methyl-L-arginine (L-DHMA). image