Journal of the American Chemical Society ARTICLE Figure 5. Substrate binding pocket. Cantilever hairpin residues and residues interacting with the aliphatic portion of R-HPP (L144, L193, and F182) are shown as sticks. Iron is shown as a sphere (rust). species,15 here we ﬁnd that the oxygen atom of NO is far from the terminal amine of Lys23 (∼4.8 Å in monomer A and ∼5.2 Å in the monomer B), although movement of this residue is possible. Alternatively, Lys23 may play its essential role15 at some other stage in the mechanism. Water Binding Site Trans to His180. Many of the HppE structures that have bound substrate also have highly ordered water molecules in the active site. Of particular interest are water molecules, which are found in the R-HPP HppE structures and lie near Lys23 in a position trans to facial triad ligand His180 without coordinating the metal (Figure 2a,b). A water molecule was observed in a similar position in one of three active sites within the asymmetric unit of the previously determined S-HPP Fe(II) HppE structure in tetragonal space group15 (Figure 2d). There is a range of distances that waters can occupy in this general water binding region (2.5 3.9 Å). Waters located at approximately 2.8 Å from the metal are in the same position as the terminal oxygen of NO when it is bound in the open metal coordination site (comparable to the second oxygen of diox- ygen) (Figure 2b,d; also see Supporting Information Figure 1e,i). Waters at distances of ∼3.9 Å from the metal may represent a product water position (Figures 2a; also see Supporting Informa- tion Figure 1a,b). Comparison of Bidentate R- and S-HPP Binding. With experimental information from our NO data supporting the assignment of the binding site for dioxygen on iron, we can examine the proposed mechanisms for regiospecificity by com- paring our S-HPP and R-HPP bound structures. Both enantio- mers bind to the same general site on the metal in the active site of HppE. For S-HPP, its hydrophobic portion including the C2 methyl group is accommodated by a hydrophobic pocket on the enzyme, comprising residues Phe182, Leu193, and Leu144 (Figure 5).15 Because the position of the C2 methyl moiety of the R-HPP molecule (bidentate binding mode) is within 1 Å of the corresponding position of the C2 methyl moiety of S-HPP (Figure 6a), the same hydrophobic binding pocket serves to accommodate the methyl moiety of the R-enantiomer (Figure 5). Even though the binding modes for both substrate enantio- mers are similar, they are not identical (Figure 6a), and even small diﬀerences in orientations of C1 and C2 can have a signiﬁcant impact in terms of accessibility of their hydrogens for abstraction by the reactive iron oxygen intermediates. While hydrogen atoms are not discernible in electron density, the orientation of the sp3 carbon atoms (i.e., tetrahedral carbon atoms) can be accurately modeled within the electron density. Therefore, the positions of the hydrogen atoms covalently bound to these carbon atoms can be inferred. Using the binding site of Figure 6. Structural insights into HppE regiospeciﬁcity. (a) Stereoview comparing a 2Fo Fc omit map density (contoured at 1.0 σ) of S-HPP (cyan carbons, blue mesh) and R-HPP (green carbons, green mesh) from the Co(II) HppE structures. (b) 2Fo Fc omit map density (left) and cartoon (right) for S-HPP, with accessible hydrogen atom in blue and inaccessible hydrogens in red. (c) 2Fo Fc omit map density (left) and cartoon (right) for R-HPP. Hydrogen atoms labeled as in (b). (d) Left, superposition of R-HPP (carbons in green) with a hypothetical conformation (not observed) of R-HPP (carbons in wheat) in omit map density for R-HPP. Right, chemical structure of R-HPP shown in a hypothetical conformation (not observed) shown as in (d), left. Accessible hydrogen in blue, and arrow indicates predicted distance. Position of NO from the NO S-HPP Fe(II) HppE structure is shown in (b) (d) for reference. NO to mark the likely location of the dioxygen binding site, the modeling of hydrogens suggests that for the S-HPP substrate, the Pro-R hydrogen at C1 points toward this putative dioxygen binding site (3.5 Å between O of NO and C1), while the C2 hydrogen is oriented in the opposite direction (4.3 Å between O of NO and C2) (Figure 6b). In contrast, for the R-HPP structures, the modeled C1 methylene hydrogens point away from (4.0 Å between O of NO and C1) while the C2 hydrogen points toward the dioxygen binding site (3.1 Å between O of NO and C2) (Figure 6c). Thus, despite the similar bidentate binding modes, the orientation of the C1 and C2 carbons and their hydrogen atoms appear to be quite diﬀerent. 11266 dx.doi.org/10.1021/ja2025728 |J. Am. Chem. Soc. 2011, 133, 11262–11269