Mechanism of the Dinuclear Iron Enzyme p‐Aminobenzoate N‐oxygenase from Density Functional Calculations

Abstract

AurF is a diiron enzyme that utilizes two dioxygen molecules as the oxidant to catalyze the oxidation of p‐aminobenzoate to p‐nitrobenzoate. Density functional calculations were performed to elucidate the reaction mechanism of this enzyme. Two different models were considered, with the oxygenated intermediate being a diferric peroxo species or a diferric hydroperoxo species. The calculations strongly favor the model with a diferric peroxo species and support the mechanism proposed by Bollinger and co‐workers. The reaction starts with the binding of a dioxygen molecule to the diferrous center to generate a diferric peroxide complex. This is followed by the cleavage of the O−O bond, concertedly with the formation of the first N−O bond, which has a barrier of only 9.2 kcal/mol. Subsequently, the first‐shell ligand Glu227 abstracts a proton from the substrate. After the delivery of two electrons from the external reductant and two protons from solution, a water molecule and the experimentally suggested intermediate p‐hydroxylaminobenzoate are produced and the diferrous center is regenerated. The oxidation of the p‐hydroxylaminobenzoate intermediate requires the binding of a second dioxygen molecule to the diferrous center to generate the diferric peroxide complex. Similarly to the oxidation of p‐aminobenzoate, the O−O bond cleavage and the formation of the second N−O bond take place in a concerted step. The p‐nitrobenzoate product is formed after the release of two protons and two electrons from the substrate. The model with a hydroperoxo species gave a much high barrier of 28.7 kcal/mol for the substrate oxidation due to the large energy penalty for the generation of the active hydroperoxo species.

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

“…Such butterfly-like structure of P1 is analogous to those proposed in sMMO, 5,94 epoxidase BoxB, 25,50 ToMo, 51,95 CmlA, 52 and AurF. 49 For the end-on binding Z-like P2, its energy is 1.8 kcal/mol higher than that of P1. In the case in which Asp315 is bidentate-coordinated to both irons, the corresponding energy of P3 is 3.8 kcal/mol higher than that of P1.…”

“…However, the subsequent work by Liao and co-workers presented an alternative mechanism for AurF. 49 Based on the DFT model calculations, it was found that N-oxygenation by the P intermediate is kinetically favored over that via the protonated P. 49 In spite of all of the aforementioned mechanistic insights, the structure and the reactivity of the peroxo-Fe 2 (III/III) intermediate in SznF are still elusive. Scheme 1 shows the putative mechanisms for the reactions of the peroxo-Fe 2 (III/ III) intermediate that emerge from previous investigations of SznF.…”

“…32 In addition to the above two mechanistic pathways, one plausible mechanism involves the electrophilic attack of peroxo species onto the N site of guanidium, which may trigger the heterolytic cleavage of the O−O bond, leading to L-HMA (Scheme 1c). Such a mechanism in Scheme 1c is reminiscent of O 2 activation mechanisms by the diiron enzymes AurF, 49 BoxB, 50 ToMo, 51 and CmlA. 52 To address the catalytic mechanism of the HDO domain in SznF, we have performed combined classical molecular dynamics (MD) simulations and hybrid QM/MM calculations.…”

QM/MM Calculations Suggest Concerted O−O Bond Cleavage and Substrate Oxidation by Nonheme Diiron Toluene/o‐Xylene Monooxygenase

“…For the concerted mechanism, the O−O bond cleavage and the electrophilic attack on the toluene substrate take place, affording a cationic tetrahedral σ ‐complex Int1 with barriers of 16.8 kcal/mol for both ortho and meta attacks. This mechanism avoids the formation of the high‐valent diferryl Q species, and this strategy has also been applied in BoxB [11a] and AurF [8f,15b,17] . Unlike BoxB, which favors the formation of epoxide products, [11b] ToMOH is reluctant to undergo epoxide formation due to the formation of a stable arenium cation intermediate.…”

Computational Study of the C5-Hydroxylation Mechanism Catalyzed by the Diiron Monooxygenase PtmU3 as Part of the Platensimycin Biosynthesis

“…In this work, based on the recently obtained crystal structure of PtmU3 in complex with the substrate, a series of computational models with different coordination modes of dioxygen as well as different reactive species (Fe2 III –superoxo or Fe2 IV –oxo) to trigger the H-abstraction have been constructed, and the successive C5 β-hydroxylation by the diiron center has been explored by using the QM/MM approach, which has been widely applied in exploring the catalytic mechanism of enzymes, including some diiron-containing enzymes. ,, …”