Journal of the American Chemical Society be invoked to explain why R-HPP forms a ketone rather than an epoxide product. Our structural data suggest that the formation of diﬀerent products is not due to the use of diﬀerential binding sites on the enzyme, but rather it is due to the conformation that the substrate itself must assume such that it ﬁts well into the same binding site in the enzyme. For R-HPP to bind in a bidentate mode to Fe, utilizing the same binding pocket on the enzyme, adjustments must be made, but here it is the substrate rather than the enzyme that adjusts. R-HPP adopts an extended and stag- gered conformation that places the phosphonate moiety as far as possible from the methyl group (Figure 6, also see Supporting Information Figure 2). The result of this conformational diﬀer- ence is that the C2 hydrogen is now closer to the dioxygen binding site than either C1 hydrogen, providing a structural rather than a chemical rationale for the observed regiospeciﬁcity in HppE catalysis. These ﬁndings are consistent with the ﬂuoride analogue data,22 which suggest that abstraction of a C1 hydrogen is involved in epoxide formation from S-HPP, but not in ketone formation from R-HPP (Scheme 1). For S-HPP, abstraction of the C1 pro-R hydrogen by the iron superoxo intermediate (Scheme 2a) would yield a substrate radical centered at C1, which could collapse into the epoxide product. For R-HPP, hydrogen atom abstraction from C2 would lead to a C2-centered radical intermediate, which could collapse into the ketone product, 2-oxo-propylphosphonic acid (Scheme 2b). ’ CONCLUSIONS This series of structures has allowed us to probe the mecha- nism of regiospeciﬁcity for HppE, a fascinating variant in the nonheme mononuclear iron family of enzymes. We now have structural evidence for the manner in which both the S- and the R-enantiomers of the substrate bind, as well as for the location of dioxygen binding to Fe. This ﬁrst three-dimensional visualization of enantiomeric substrate recognition provides new insight into the stereospeciﬁcity aﬀorded by a mononuclear nonheme iron enzyme. ’ ASSOCIATED CONTENT bS Supporting Information. Two-dimensional active site contact maps (Figure 1a i), Newman projections of substrates bound to iron (Figure 2), data collection and reﬁnement statistics table (Table 1), and structures in paper table (Table 2). This material is available free of charge via the Internet at http://pubs. acs.org. The structures are deposited in the Protein Data Bank with PDB entries 3SCF, 3SCG, and 3SCH. ’ AUTHOR INFORMATION Corresponding Author cdrennan@mit.edu Present Addresses ^Harbor Hospital, 3001 South Hanover Street, Baltimore, Mary- land 21225, United States. ’ ACKNOWLEDGMENT This work was supported in part by National Institutes of Health Grants GM40541 (HWL), F32 GM079966 (DY), and Center Grant P30 ES002109. C.L.D. is a Howard Hughes Medical Institute Investigator. Data were collected at the ARTICLE Advanced Light Source (ALS), Stanford Synchrotron Radiation Laboratory (SSRL), and the Advanced Photon Source (APS). Support for the synchrotron sources is provided by the U.S. Department of Energy (ALS, SSRL, and APS) and by the National Institutes of Health (SSRL). ’ REFERENCES (1) Lobel, B. Int. J. Antimicrob. Agents 2003, 22, 85–87. (2) Nakazawa, H.; Kikuchi, Y.; Honda, T.; Isago, T.; Nozaki, M. J. 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