Journal of the American Chemical Society Scheme 1. HppE-Catalyzed Conversion of (a) S-HPP, (b) R-HPP, (c) S-FHPP, and (d) R-FHPP Figure 1. The HppE tetramer is shown colored by molecule in blue, magenta, yellow, and green, with strand 1 and the cantilever hairpin colored separately in cyan, pink, wheat, and limon, respectively. Iron atoms are shown as spheres (rust), and iron ligands are in sticks: carbon (gray), nitrogen (blue), oxygen (red). centered at C1 would then yield the epoxide product, whereas the C2 radical yields the ketone.22 Although the results from these ﬂuorinated analogues provide an explanation for why a diﬀerent product is formed from each stereoisomer, we sought to understand why the enzyme would abstract a diﬀerent hydrogen atom in each case. First, it is important to identify where dioxygen binds to the iron in the enzyme active site. While it has been proposed that dioxygen binds iron at the only open coordination site that exists when S-HPP is bound in the bidentate conformation,15 here we use NO as a dioxygen mimic to obtain experimental evidence for this binding site. The next question is whether the R- and S-stereo- isomers bind the iron active site in a diﬀerent fashion, and, if so, ARTICLE what is the cause of diﬀerential binding? If they bind in the same fashion, can diﬀerences in C H bond dissociation energies (calculated to be 96.5 and 89.0 kcal mol 1 for S-HPP (C1 H) and R-HPP (C2 H), respectively6) explain the observed re- giospeciﬁcity? To address these questions, we have solved a structure at 2.1 Å resolution of Co(II) HppE with the R-stereoisomer of substrate bound. Additionally, we have deter- mined the structure of the substrate enantiomer in complex with Fe(II) HppE at 3.0 Å resolution. These structures reveal that R-HPP has a binding mode similar to that of S-HPP but diﬀers with respect to the relative orientation of the C1, C2, and C3 positions. Furthermore, the structures illustrate that hydrogen atom accessibility to the proposed iron-superoxo intermediate species is suﬃcient to explain the regiospeciﬁcity of the HppE- catalyzed reactions. ’ MATERIALS AND METHODS Materials. R-HPP and S-HPP were synthesized as previously described.9,10 Cobalt(II) chloride was purchased from Hampton Re- search (Aliso Viejo, CA), diethylamine NONOate was from Cayman Chemical (Ann Arbor, MI), and other reagents were from Sigma-Aldrich (St. Louis, MO). Protein Isolation and Enzymatic Assays. Wild-type and selenomethione-derivatized apo-HppE was expressed and purified as previously described.10,15 Apo-protein was concentrated to 30 mg/mL and dialyzed into 0.02 M Tris-HCl, at pH 8.0. Enzyme activity of apo- HppE reconstituted with Fe(II)(NH4)2(SO4)2 3 6H2O was determined using a 31P NMR assay.9,10 Crystallization of R-HPP Fe(II) HppE and R-HPP Co(II) HppE. R-HPP Fe(II) HppE crystals were obtained using previous anaerobic conditions for S-HPP Fe(II) HppE crystals15 replacing the substrate S-HPP with its enantiomer. R-HPP (0.6 μL, 0.2 M) was added to Fe(II) HppE crystals placed over a reservoir of soaking buffer solution (0.1 M HEPES, pH 7.5, 2.5 M ammonium sulfate) and allowed to diffuse into the crystals for 14 h. The crystals were flash- cooled in liquid nitrogen following a 30 s transfer to a cryo-protectant buffer solution (0.1 M Tris-HCl, pH 8.0, 2.0 M ammonium sulfate, 30% (w/v) xylitol). Crystals of R-HPP in complex with Co(II) HppE were obtained by soaking this substrate into Co(II) HppE crystals.15 The hexagonal crystals used for soaking were grown by mixing 2.0 μL of 30 mg/mL selenomethionine apo-HppE solution in 0.02 M Tris HCl pH 8.0 with 2.0 μL of precipitant buﬀer solution (0.1 M Tris HCl, pH 8.5, 2.0 M ammonium sulfate) using the hanging-drop vapor diﬀusion method at room temperature. CoCl2 (0.3 μL, 0.1 M) solution was added to the drop, and the so-called “Tris Co(II) HppE” crystals grew in 24 h. R-HPP Co(II) HppE crystals were obtained from Tris Co(II) HppE crystals by ﬁrst transferring crystals to soaking buﬀer solution to displace any Tris molecules coordinating Co(II). The removal of Tris was veriﬁed through a structure determination (data not shown). The Co(II) HppE crystals were transferred to 4.0 μL of fresh soaking buﬀer solution for 10 min. R-HPP (1.0 μL, 2.5 M) was added to the drop, and the resulting solution was placed over a reservoir of soaking solution for additional 14 h. The crystals were transferred to cryo- protectant buﬀer solution for 30 s and cooled in liquid nitrogen. Crystallization of S-HPP Fe(II) HppE NO. The ternary complex (S-HPP Fe(II) HppE) was crystallized anaerobically as previously described15 with slight modifications. S-HPP (0.6 μL, 0.2 M) was added to Fe(II) HppE crystals placed over a reservoir of soaking buffer solution. Nitric oxide (NO) was introduced to the well with S-HPP Fe(II) HppE crystals by liberation from diethylamine NONOate.23,24 After 1 h of equilibration, NO gas (2.0 mL) was injected 11263 dx.doi.org/10.1021/ja2025728 |J. Am. Chem. Soc. 2011, 133, 11262–11269