Peroxo-Diiron(III/III) as the Reactive Intermediate for N-Hydroxylation Reactions in the Multidomain Metalloenzyme SznF: Evidence from Molecular Dynamics and Quantum Mechanical/Molecular Mechanical Calculations

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 Nω-methyl-l-arginine (l-NMA) to generate Nδ-hydroxy-Nω-methyl-l-Arg (l-HMA) and Nδ,Nω-dihydroxy-Nω,-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.

Computational Exploration of Enzyme Promiscuity: Mechanisms of O2 and NO Reduction Activities of the Desulfovibrio gigas Flavodiiron Protein

“…Conversely, the spin populations on the two oxygen atoms are relatively small, measuring 0.25 and −0.26, respectively (Figure S7). Therefore, the electronic structure of React OR is interpreted as featuring an antiferromagnetic coupling between two high-spin ferric ions. ,,− These results also strongly indicate that upon O 2 binding to the diiron center, each ferrous ion donates one electron to the two degenerate π* orbitals of the O 2 moiety, which leads to the formation of a peroxide species with the O–O bond distance of 1.40 Å. Because of the hydrogen bond interactions between the O 2 moiety and the adjacent tyrosine residues (Tyr26 and Tyr193), it is slightly longer than the distance of the O–O bond (around 1.32 Å) in the suggested stable μ-η 1 :η 1 peroxide intermediate in cyanobacterial aldehyde-deformylating oxygenase and acyl carrier protein Δ 9 -desaturase …”

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

“…Very recently, Wang et al. proposed a mechanism, [22] elucidated through QM/MM calculations, where a butterfly‐like μ‐η 2 :η 2 ‐peroxo diiron(III/III) species acts as the starting species, which attacks the substrate's guanidium, leading to the heterolytic O−O bond cleavage. Subsequently, the hydroxylation is completed through the proton transfer from the N1 site to Glu281 and then to the O connected to the substrate, with a rate‐limiting energy barrier of 20.0 kcal/mol.…”

N-Hydroxylation and Hydrolysis by the DnfA/B/C Multienzyme System Involved in the Aerobic N2 Formation Process

“…DnfA had the highest identity (28%) with CmlI (the nonheme di-iron N -oxygenase), implying that its function is N -oxygenation. − N -Oxygenases such as CmlI, PrnD, and AurF all oxidize an amino group (−NH 2 ) to −NHOH and nitro groups (N valence state, +3). − In contrast, DnfA exhibited N -oxygenation activity toward the amide-NH 2 of glutamine to generate an N -hydroxyl group, illustrating its indispensable role in N 2 (N valence state, 0) production from −3 valence state-N via a novel oxidation mechanism. To the best of our knowledge, this is the first reported oxygenase capable of converting a fully reduced amino group (−NH 2 ) into an N -hydroxyl group and to N 2 under aerobic conditions.…”

“…37−39 New Noxygenases, such as SznF in streptozotocin biosynthesis, have been recently identified as well. 40,41 However, they all oxidize the amino group to −NHOH and nitro groups (N valence state, +3), and little is known regarding the conversion of amino groups to N 2 groups. DnfA showed homology with CmlI and AurF, suggesting that DnfA might oxidize amino group and be indispensable in N 2 (N valence state, 0) production via a novel oxidation mechanism.…”

“…Oxygenases are widely distributed enzymes capable of integrating molecular oxygen into biomolecules. − Several types of N -oxygenases have been reported: PrnD involved in the pyrrolnitrin biosynthesis pathway in Pseudomonas fluorescens, AurF in the aureothin biosynthesis pathway in Streptomyces thioluteus, , and CmlI in the chloramphenicol biosynthesis pathway in Streptomyces venezuelae. − New N -oxygenases, such as SznF in streptozotocin biosynthesis, have been recently identified as well. , However, they all oxidize the amino group to −NHOH and nitro groups (N valence state, +3), and little is known regarding the conversion of amino groups to N 2 groups. DnfA showed homology with CmlI and AurF, suggesting that DnfA might oxidize amino group and be indispensable in N 2 (N valence state, 0) production via a novel oxidation mechanism.…”