10.1021/jp809838k.s001 Muhannad Altarsha Muhannad Altarsha Dongqi Wang Dongqi Wang Tobias Benighaus Tobias Benighaus Devesh Kumar Devesh Kumar Walter Thiel Walter Thiel QM/MM Study of the Second Proton Transfer in the Catalytic Cycle of the D251N Mutant of Cytochrome P450cam American Chemical Society 2009 cytochrome P 450cam proton transfer proton transfer mechanisms MD D 251N models Asn 251 residue Second Proton Transfer model III Cytochrome P 450camProtonation D 251N mutation D 251N uncoupling reaction D 251N Mutant QM 2009-07-16 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/QM_MM_Study_of_the_Second_Proton_Transfer_in_the_Catalytic_Cycle_of_the_D251N_Mutant_of_Cytochrome_P450cam/2842699 Protonation of Compound 0 in the catalytic cycle of cytochrome P450cam may lead to the formation of either the reactive Compound I (coupling) or the ferric resting state (uncoupling). In this work, we investigate the effect of the D251N mutation on the coupling and uncoupling reaction by combined quantum mechanics/molecular mechanics (QM/MM) calculations. The mutated Asn251 residue has two possible orientations, i.e. directed toward the active site (no flip) or away from the active site (flip), with the latter one being preferred in classical molecular dynamics (MD) simulations. The possible proton transfer mechanisms in the coupling and uncoupling reaction were studied for three models of the D251N mutant, i.e. no flip (model I), flip (model II), and flip with an extra water (model III). According to the QM/MM calculations, the uncoupling reaction is always less favorable than the coupling reaction. The coupling reaction in the D251N mutant follows the same mechanism as in the wild-type enzyme, with initial O−O cleavage followed by proton transfer. The barrier for the initial step is similar in all D251N models, but the proton transfer is most facile in model III. The hydroxide anion formed in model III is not reprotonated easily by neighboring residues, while proton delivery from bulk solvent seems possible via a water network that remains intact during 2 ns classical MD simulation. The computational results are consistent with the experimental findings that the coupling reaction dominates the consumption of dioxygen in the D251N mutant, but with lower activity than in the wild-type enzyme.