Protein Free Energy Corrections in ONIOM QM:MM Modeling: A Case Study for Isopenicillin N Synthase (IPNS)

The protein environment can have significant effects on the enzyme catalysis even though the reaction occurs locally at the reaction center. In this paper, we describe an efficient scheme that includes a classical molecular dynamics (MD) free-energy perturbation (FEP) correction to the reaction energy diagram, as a complement to the protein effect obtained from static ONIOM (QM:MM) calculations. The method is applied to eight different reaction steps, from the O-2-bound reactant to formation of a high-valent ferryl-oxo intermediate, in the nonheme iron enzyme isopenicillin N synthase (IPNS), for which the QM:MM energy diagram has previously been computed [Lundberg, M. et al. J. Chem. Theory Comput. 2009, 5, 220-234]. This large span of the reaction coordinate is covered by dividing each reaction step into microsteps using a virtual reaction coordinate, thus only requiring ONIOM information about the stationary points themselves. Protein effects are important for C-H bond activation and heterolytic O-O bond cleavage because both these two steps involve charge transfer, and compared to a static QM: MM energies, the dynamics of the protein environment changes the barrier for O-O bond cleavage by several kcal/mol. The origin of the dynamical contribution is analyzed in two terms, the geometrical effect caused by the change in average protein geometry (compared to the optimized geometry) in the room temperature MD simulation with the solvent, and the statistical (entropic) effect resulting from fluctuations in the interactions between the active site and the protein environment. These two effects give significant contributions in different steps of the reaction.