Journal of the American Chemical Society Scheme 2. Proposed Mechanisms for the Oxidation of S- and R-HPP by HppEa ARTICLE a (a) Conversion of S-HPP to fosfomycin is initiated by H-atom abstraction from C1. (b) Conversion of R-HPP to the keto-product probably occurs via H-atom abstraction from C2. It is interesting to consider why R-HPP binds to the iron with such a diﬀerence in the positions of C1 and C2 as compared to S-HPP, and whether at this resolution we can be sure of this alternative conformation. When we compare the reﬁned model of R-HPP (green in Figure 6c,d) with a hypothetical conforma- tion of R-HPP that is based on the S-substrate conformation (wheat in Figure 6d), we ﬁnd that there is no electron density to support the placement of the methyl group on C2 in the con- formation represented by the hypothetic model (Figure 6d). The alternative conformation is a much better ﬁt to the density. In considering why the hypothetical conformation is not favored, modeling suggests that the methyl group on C2 would be close (2.8 Å) to the phosphonic acid moiety in this conformation, producing a steric clash (Figure 6d). The electron density is consistent with an extended and staggered conformation of R-HPP in which the methyl moiety is farthest from the bulky phosphonic acid moiety (Figure 6c; also see Supporting Infor- mation Figure 2). ’ DISCUSSION Mononuclear iron enzymes catalyze a wide range of reactions from antibiotic biosynthesis to DNA repair. While the products of the reactions vary, most mononuclear iron enzymes must accomplish the same tasks (activate the iron center to facilitate dioxygen binding, protect high energy intermediates throughout the catalytic cycle, and reduce dioxygen).9,10,12,15,21,33 Our goal with these structural analyses is to address the directing factors by which HppE converts each substrate to its ﬁnal product, provid- ing insight into the mechanisms of regiospeciﬁcity. For the mechanism of the enzyme with its natural substrate, S-HPP, much is already known, and here we provide structural insight into dioxygen binding. For R-HPP, no structural data were available, and we wished to obtain crystallographic snap- shots for the stages of catalysis and compare these results to those for S-HPP. First, in considering how the Fe(II/III) potential is modulated in HppE to favor oxidation by dioxygen, our previous snapshots of S-HPP HppE suggested that direct bidentate binding of the electron-donating substrate molecule S-HPP to Fe(II) would be suﬃcient to activate the iron center.15 We now observe that R-HPP also binds to iron in a bidentate manner via its phosphonate and hydroxyl oxygen atoms (Figure 2). Second, we previously considered how reactive intermediates are pro- tected in the HppE active site when S-HPP is the substrate. We identiﬁed an induced ﬁt mechanism in which a switch in the S-HPP binding mode from monodentate to bidentate induces a conformational change of two β-strands that seals oﬀ part of the active site, protecting reactive catalytic intermediates.15 Interest- ingly, this induced ﬁt mechanism is in play with R-HPP as well (Figure 3). Because the two enantiomeric substrates use the same coordination sites for binding to iron and both use the same binding pocket in the enzyme (Figures 2 and 5), both substrates are able to induce the same conformational change in the β-hairpin upon bidentate substrate binding. While much of the active site is buried from solvent due to this conformational change, there is a small channel available for dioxygen to enter and reach the only open coordination site on Fe in the S-HPP bound HppE structures.15 We have proposed that this site is the location of dioxygen binding.15 Here, we use NO as a mimic for dioxygen binding and conﬁrm that NO does bind to this open coordination site (Figure 4), providing the ﬁrst structural evidence in support of this hypothesis. The binding of dioxygen to the Fe(II) center is proposed to lead to the reversible formation of a transient Fe(III) superoxo species9,21 and facil- itate hydrogen atom abstraction from S-HPP (Scheme 2a).20,34 In the ﬁnal steps of the mechanism, the substrate radical intermediate collapses, forming the epoxide product fosfomycin, while dioxygen is fully reduced in the presence of NAD(P)H, forming two water molecules (Scheme 2a). One water molecule may remain bound to iron until it is displaced by the binding of a second substrate molecule, consistent with the observation of multiple water molecules bound to iron in the absence of substrate.15 The second water molecule would be released from iron and may occupy the farther water binding site (3.9 Å), trans to His180 as described above. While this series of structures nicely explains the observed reaction of S-HPP with HppE to form an epoxide, so far this discussion has only noted similarities between S-HPP and R-HPP binding and has not described any diﬀerences that can 11267 dx.doi.org/10.1021/ja2025728 |J. Am. Chem. Soc. 2011, 133, 11262–11269