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 p K a values, which are considered to be a crucial factor for theoretically exploring the reaction rhythm. The estimated p K 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 –O 2 •– 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.