The superoxide reductase is an enzyme catalyzing the reduction of superoxide (O2.-) into milder hydrogen peroxide (H2O2). It includes a non-heme iron active site (Fe-His4Cys, SOR) where the reduction occurs and a desulforedoxin-like center (Fe-Cys4, DX). The catalytic cycle exhibits two protonations with possible involvement of the lysine nearby. The first one occurs on the distal oxygen and has already been theoretically studied. In this study, we focus on the mechanism of the second protonation on the proximal oxygen to form H2O2 in the wild type and expend the resulting methodology to simulate and explain the reactivity of mutants. To achieve this goal, we have developed Amber force field parameters for the SOR and DX site. Their robustness were evaluated and they were used to report the hydrogen bonding network around the active site. Then, we study the 2nd protonation step using well-tempered QM/MM molecular metadynamics simulations within CP2K package. Due to the high spin nature of the active iron, the XC functional employed here is B3LYP yielding a correct spin state compared to standard GGA. Thus, we present here a reliable method to simulate enzyme catalysis activity with a hybrid DFT in ab-initio MD avoiding the need of empirical Hubbard parameter (LDA+U). Result of metadynamics shows a cumulative barrier of approximately 15 kcal.mol-1, in agreement with experiment. The lysine residue at the entrance of the active site serves in the wild type as the proton source either directly or involving bridging water molecule via proton hopping. Acknowledgements: LABEX ARCANE ANR-11-LABX-0003-01 & CIMENT MESOCENTRE GRENOBLE.

Understanding the formation of hydrogen peroxyde in superoxide reductase: A metadynamic QM/MM study

251st National Meeting (2016)

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