Computational Insights into the N-Migration and Oxidative Rearrangement Involved in the N -Nitrosourea Formation Catalyzed by the Cupin Domain of Multidomain Metalloenzyme SznF

The multidomain metalloenzyme SznF can specifically catalyze the conversion of N^ω-methyl-l-arginine (l-NMA) to N^δ-hydroxy-N^ω-methyl-N^ω-nitroso-l-citrulline (l-NHMA), which is the key step for the biosynthesis of the N-nitrosourea pharmacophore, a precursor to the pancreatic cancer drug streptozotocin (SZN). The central domain of SznF is responsible for mediating the two sequential hydroxylations of l-NMA at N^δ and N^ω positions to first generate N^δ,N^ω-dihydroxy-N^ω-methyl-l-arginine (l-DHMA), and the cupin domain of SznF promotes the N-migration and oxidative rearrangement of l-DHMA. This structural rearrangement contains both the C═N bond cleavage and N-N bond formation, and it is very challenging for chemical synthesis. To illuminate the catalytic mechanism of the cupin domain of SznF, we constructed the reactant models and performed a series of QM/MM calculations. We first determined the protonated states of two hydroxyls and imino of l-DHMA by calculating their pK_a values, which are considered to be a crucial factor for theoretically exploring the reaction rhythm. The estimated pK_a values revealed that the two hydroxyls and imino of l-DHMA should be in protonated states, and the previously proposed reaction mechanism in which superoxo addition to the unsaturated carbon as the first step is unlikely. Instead, the Fe^II-O₂^•- unit should first abstract a hydrogen from the N^ω-hydroxyl group to trigger the reaction, and then the generated Fe^III-OOH attacks the unsaturated carbon to form the peroxide-bridged intermediate, followed by the concerted O-O and N-C bond cleavage leading to the formation of the Fe-coordinated NO radical, which is the precondition for N-migration. During the reaction, the iron ion plays important roles, not only as a central ion to coordinate with the substrate to mediate the H-abstraction, Fe-OOH attack as well as the bond cleavage and formation but also in stabilizing the NO radical and promoting the final N-N bond formation. These results may deepen the understanding of the catalysis of nonheme iron enzymes.