Iron Hydroperoxide Intermediate in Superoxide Reductase: Protonation or Dissociation First? MM Dynamics and QM/MM Metadynamics Study

Abstract

Superoxide reductase is a mononuclear iron enzyme involved in superoxide radical detoxification in some bacteria. Its catalytic mechanism is associated with the remarkable formation of a ferric hydroperoxide Fe-OOH intermediate, which is specifically protonated on its proximal oxygen to generate the reaction product HO. Here, we present a computational study of the protonation mechanism of the Fe-OOH intermediate, at different levels of theory. This was performed on the whole system (solvated protein) using well-tempered metadynamics at the QM/MM (B3LYP/AmberFF99SB) level. Enabled by the development of a new set of force field parameters for the active site, a conformational MM study of the Fe-OOH species gave insights into its solvation pattern, in addition to generating the two starting conformations for the ab initio metadynamics setup. Two different protonation mechanisms for the Fe-OOH intermediate have been found depending on the starting structure. Whereas a possible mechanism involves at first the protonation of the hydroperoxide ligand and then dissociation of HO, the most probable one starts with an unexpected dissociation of the HOO ligand from the iron, followed by its protonation. This favored reactivity was specifically linked to the influence of both the nearby conserved lysine 48 residue and the microsolvatation on the charge distribution of the oxygens of the HOO ligand. These data highlight the crucial role of the whole environment, solvent, and protein, to describe accurately this second protonation step in superoxide reductase. This is clearly not possible with smaller models unable to reproduce correctly the mechanistically determinant charge distribution.

Conformational H-bonding modulation of the iron active site cysteine ligand of superoxide reductase: absorption and resonance Raman studies

“…The catalytic cycle of SORs has been extensively studied, both experimentally [24][25][26][27] and theoretically. [28][29][30] All the data agree with an inner sphere reduction mechanism, involving O2 •binding at the free sixth coordination position of the ferrous center as first step. This reaction is extremely fast and together with a single protonation process, the reduction of O2 •leads to the formation of a high-spin ferric iron hydroperoxide intermediate (Fe 3+ -OOH).…”

Insights into the Structures of Superoxide Reductases from the Symbionts Ignicoccus hospitalis and Nanoarchaeum equitans

“…This residue has been proposed to contribute to attracting the anionic substrate by increasing the positive surface charge around the catalytic site 17 and providing the proton to the superoxo/hydroperoxo intermediate, directly or through a chain involving water molecules, to generate the product, H 2 O 2 . 15,18,19 The function of these two residues has been addressed by enzymatic studies of several SOR site-directed mutants, 17,20−22 and of "natural" mutants, i.e., SORs lacking either the glutamate (from Nanoarchaeum equitans 23 ) or the lysine (from Ignicoccus hospitalis 24 ). These studies showed that in vitro the absence of the glutamate does not have any consequence in catalysis, while the mutation of the lysine led to a decrease in the rate constant for the formation of the first catalytic intermediate, from 1 × 10 9 M −1 s −1 for the wild-type enzyme to 4.2 × 10 7 M −1 s −1 for the lysine mutant.…”

“…X-ray and FTIR studies showed that the glutamate is involved in a redox-linked conformational change, upon reduction of the ferric iron, being detached from the metal ion with a concomitant movement of a loop that contains the glutamate and lysine residues. ,, Simultaneously, the lysine, which is ∼12 Å from the metal in the ferric form, approaches the reduced metal ion. This residue has been proposed to contribute to attracting the anionic substrate by increasing the positive surface charge around the catalytic site and providing the proton to the superoxo/hydroperoxo intermediate, directly or through a chain involving water molecules, to generate the product, H 2 O 2 . ,, …”

Analytical hessian fitting schemes for efficient determination of force‐constant parameters in molecular mechanics

“…This is due partly to the growing interest in MM‐ and QM/MM‐type computer simulations of nonbiological molecular systems such as metal–organic frameworks (MOFs), which could sometimes go beyond the chemical space covered by existing parameter sets. In addition, even for biological systems, parameterization is necessary when a metal ion or some other cofactor is involved …”