N-Nitrosation Mechanism Catalyzed by Non-heme Iron-Containing Enzyme SznF Involving Intramolecular Oxidative Rearrangement

Abstract

The non-heme iron-dependent enzyme SznF catalyzes a critical N-nitrosation step during the N-nitrosourea pharmacophore biosynthesis in streptozotocin. The intramolecular oxidative rearrangement process is known to proceed at the Fe IIcontaining active site in the cupin domain of SznF, but its mechanism has not been elucidated to date. In this study, based on the density functional theory calculations, a unique mechanism was proposed for the N-nitrosation reaction catalyzed by SznF in which a four-electron oxidation process is accomplished through a series of complicated electron transferring between the iron center and substrate to bypass the high-valent Fe IV O species. In the catalytic reaction pathway, the O 2 binds to the iron center and attacks on the substrate to form the peroxo bridge intermediate by obtaining two electrons from the substrate exclusively. Then, instead of cleaving the peroxo bridge, the C ε −N ω bond of the substrate is homolytically cleaved first to form a carbocation intermediate, which polarizes the peroxo bridge and promotes its heterolysis. After O−O bond cleavage, the following reaction steps proceed effortlessly so that the N-nitrosation is accomplished without NO exchange among reaction species.

Dioxygen Activation and Nδ,Nε-Dihydroxylation Mechanism Involved in the Formation of N-Nitrosourea Pharmacophore in Streptozotocin Catalyzed by Nonheme Diiron Enzyme SznF

“…At present, the mechanism of N-nitrosation reaction catalyzed by the cupin domain of SznF has been suggested on the basis of the X-ray crystal structure (PDB: 6M9R). 4,5 However, the mechanism of two successive N-hydroxylation reactions of L-NMA catalyzed by SznF in the central domain is still unclear. The X-ray structure of SznF 1 (PDB: 6M9R) revealed a mononuclear iron cofactor in the cupin domain that is facially coordinated by three histidine ligands.…”

“…1 Although the diiron(III,III)-peroxo intermediate has been previously detected in other N-oxygenases enzymes, such as AurF and CmlI, 12−17 SznF provides the first example of such an intermediate in the N-oxygenases within the heme oxygenase-like diiron oxidase superfamily. 11 Although the N-nitrosation reaction catalyzed by the cupin domain of SznF has been suggested, 4,5 dioxygen is also unknown. Herein, a combined molecular dynamics (MD) simulations and quantum mechanical/ molecular mechanical (QM/MM) calculations were carried out to elucidate the catalytic reaction details of successive Nhydroxylation reactions, including the mechanism of activating O 2 , the binding model of L-NMA, and the most likely reaction pathways.…”

“…Thus, it is of vital importance to explore the formation mechanism of SZN, in particular that of the N-nitrosourea complex formation. At present, the mechanism of N-nitrosation reaction catalyzed by the cupin domain of SznF has been suggested on the basis of the X-ray crystal structure (PDB: 6M9R). , However, the mechanism of two successive N-hydroxylation reactions of L-NMA catalyzed by SznF in the central domain is still unclear.…”

“…Although the N-nitrosation reaction catalyzed by the cupin domain of SznF has been suggested, , the details of N-hydroxylation of L-NMA are still unclear. In addition, how the diiron cofactor in the central domain of SznF activates the dioxygen is also unknown.…”

Unusual Dioxygen-Dependent Reactions Catalyzed by Nonheme Iron Enzymes in Natural Product Biosynthesis

“…216 After O 2 binds to Fe(II), a C ε −O bond may be formed to generate the peroxo bridge intermediate The key difference between the two possible mechanisms is that while the Fe(IV)−NO is developed in the first mechanism, the rearrangement in the second mechanism does not involve substrate fragmentation and the oxidation state of the iron center does not change throughout the reaction. Although the mechanistic evaluation by DFT computation suggested that the second mechanism is energetically less favorable, 222 further experimental investigations will be necessary to better understand this unusual rearrangement reaction.…”