Biochemistry Analysis of the hydrogen bonding to the epoxide oxygen atom during the ring-opening reaction revealed that lower barriers were observed when a hydrogen bond was present between the epoxide oxygen atom and the hydroxyl group of Tyr465 in the transition state. The hydrogen bond between the Tyr381 hydroxyl group and the epoxide oxygen was found to always be present during all simulations. The results also support the roles of the active site tyrosine residues as being electrophilic catalysts, rather than per- forming general acid catalysis, in agreement with experimental proposals.32 The calculations presented here support the protonated form of His523 to be present during the nucleophilic ring-opening step. Evidence of this involvement includes correct prediction of regioselectivity, lower activation free energies, and increased hydrogen bond stabilization of transition states. These features are exclusive to this protonation state (i.e., are not found when it is modeled as neutral). This work provides a further example of a case in which QM/MM modeling can provide useful insight into the mechanism of an enzyme-catalyzed reaction, highlighting important interactions involved in the stabilization of key reacting species. Consideration of possible protonation states of active site residues, and where necessary, alternative binding poses, may be necessary. We demonstrate that, as long as adequate conformational sampling is performed, reliable predictions of selectivity can be made with relatively low levels of QM theory. However, to obtain absolute values of free energy barriers to near-chemical accuracy, corrections with higher-level correlated ab initio methods are required. ■ ■ ■ ■ ASSOCIATED CONTENT *S Supporting Information Details of the system setup, free energy barriers, potential energy barriers, atomic charges, absolute energies, some bond lengths, coordinates of selected species, and hydrogen bonding information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: adrian.mulholland@bristol.ac.uk. Phone: +44 (0)117 928 9097. Fax: +44 (0)117 925 0612. Funding This work was supported by an Engineering and Physical Sciences Research Council Leadership Fellowship (A.J.M., Grant EP/G007705/1; Grant EP/J010588/1 (CCP-BioSim), A.J.M. and R.L.) the Biotechnology and Biological Sciences Research Council (06, S.H. and A.J.M.), and a European Union Quality of Life FP5 program Marie Curie Individual Fellowship (L.R., Contract QLRI-CT-1999-51244). Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS REFERENCES We thank Dr. Christopher Woods for advising in the pre- paration of the revised manuscript, Prof. Fahmi Himo for useful discussions, and the referees for useful comments. (1) Morisseau, C., and Hammock, B. D. (2005) Epoxide hydrolases: Mechanisms, inhibitor designs, and biological roles. Annu. Rev. Pharmacol. Toxicol. 45, 311−333. Article (2) Chacos, N., Capdevila, J., Falck, J. R., Manna, S., Martin-Wixtrom, C., Gill, S. S., Hammock, B. D., and Estabrook, R. W. 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