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Hydrogen peroxide is a natural oxidantthat can oxidize proteinthiols (RSH) via sulfenic acid (RSOH) and sulfinic acid (RSO2H) to sulfonic acid (RSO3H). In this paper, we study thecomplete anionic and neutral oxidation pathway from thiol to sulfonicacid. Reaction barriers and reaction free energies for all three oxidationsteps are computed, both for the isolated substrates and for the substratesin the presence of different model ligands (CH4, H2O, NH3) mimicking the enzymatic environment. Wefound for all three barriers that the anionic thiolate is more reactivethan the neutral thiol. However, the assistance of the environmentin the neutral pathway in a solvent-assisted proton-exchange (SAPE)mechanism can lower the reaction barrier noticeably. Polar ligandscan decrease the reaction barriers, whereas apolar ligands do notinfluence the barrier heights. The same holds for the reaction energies:they decrease (become more negative) in the presence of polar ligandswhereas apolar ligands do not have an influence. The consistentlynegative consecutive reaction energies for the oxidation in the anionicpathway when going from thiolate over sulfenic and sulfinic acid tosulfonic acid are in agreement with biological reversibility. [ABSTRACT FROM AUTHOR]

Recently, it was found that mutations in the binding cavity of drug-metabolizing Cytochrome P450 BM3 mutants can result in major changes in regioselectivity in testosterone (TES) hydroxylation. In the current work, we report the intrinsic reactivity of TES' C-H bonds and our attempts to rationalize experimentally observed changes in TES hydroxylation using a protein structure-based in silico approach, by setting up and employing a combined Molecular Dynamics (MD) and ligand docking approach to account for the flexibility and plasticity of BM3 mutants. Using this approach, about 100,000 TES binding poses were obtained per mutant. The predicted regioselectivity in TES hydroxylation by the mutants was found to be in disagreement with experiment. As revealed in a detailed structural analysis of the obtained docking poses, this disagreement is due to limitations in correctly scoring hydrogen-bonding and steric interactions with specific active-site residues, which could explain the experimentally observed trends in regioselectivity in TES hydroxylation.