Journal of the American Chemical Society ARTICLE Figure 4. The structure of S-HPP Fe(II) HppE in complex with NO. (a) The active site of S-HPP Fe(II) HppE is shown with the Fe(II) atom (rust) as a sphere and substrate, residues, and NO as sticks. An Fo Fc omit map (green mesh) is contoured at 7.0 σ. (b) When water is reﬁned in a position similar to that of the nitrogen of NO in the active site, shown in Figure 4a, positive Fo Fc diﬀerence density (green mesh) at 4.0 σ indicates that water is not large enough to account for the density. ’ RESULTS Structures of HppE with R-HPP. The crystal structure of Co(II) HppE in complex with R-HPP was solved to 2.1 Å resolution (Figure 2a, Supporting Information Table 1). R-HPP Co(II) HppE is catalytically inert under aerobic conditions9 and allows us to observe at high resolution an enzyme state that mimics the form of the enzyme prior to dioxygen binding. We have also solved this same structure using the native Fe(II) metal ion in the absence of dioxygen (Figure 2b), but to lower resolution (3.0 Å). We find that the overall structures are similar, with root-mean-square deviations of 0.27 0.71 Å. In each case, substrate can be unambiguously positioned in the active site due to the presence of 8 10 σ electron density peaks that identify the position of the HPP phosphorus atom (Figure 2). As was the case for the S-enantio- mer, R-HPP binds to the active site metal in a bidentate binding mode coordinated by both the C2 hydroxyl and the C1 phos- phonate moieties (Figure 2a d; also see Supporting Information Figure 1a 1e).15 The bidentate binding mode is observed in both the Fe(II) and the Co(II) structures (see Supporting Information Table 2 for a complete list of structures). Conformational Change Closing Active Site. HppE appears to use an induced-fit mechanism to protect high-energy iron oxygen species formed during catalysis.15 A β-hairpin called the cantilever hairpin (residues 90 107) responds to the physiological substrate S-HPP when bound to the enzyme in the correct orientation by moving in and sealing off the top portion of the active site, leaving only a small opening for dioxygen to enter.15 Comparison of S-HPP Fe(II) HppE structures with the alternative R-HPP substrate shows a similar response; the cantilever hairpin closes down and becomes more ordered upon bidentate substrate binding. In fact, in the closed conformation, the cantilever hairpin in the R-HPP Fe(II) HppE structure superimposes well with that in the S-HPP Fe(II) HppE structure (PDB ID 1ZZ8) (Figure 3), producing very similar interactions between the protein and S- and R-HPP substrates. A number of hydrogen bonds and electrostatic interactions made by the protein with the substrate are contributed from residues of the cantilever hairpin (Arg97 and Tyr105). Other contacts are made from other β-barrel residues as well as one residue of the helical domain (Lys23). In the bidentate con- formation of both S- and R-HPP, all hydrogen-bonding interac- tions are made to the phosphonate moiety. In every structure, the phosphonate substituent is stabilized by charge charge interactions with Lys23 from the helical domain and hydrogen-bonding interactions with Tyr105 and Asn135 of the β-barrel (Supporting Information Figure 1). In the majority of both the S-HPP and the R-HPP bound Fe-HppE structures, Arg97 is ordered and con- tacts the phosphonate moiety both directly and indirectly via a water molecule to further stabilize the additional charge in the active site (Supporting Information Figure 1c i). Structure with NO as Mimic for Dioxygen Binding. With the hairpin closed, there is space available for oxygen to enter the active site in a cavity created at the interface of the helical and β-barrel domains.15 This cavity leads to the only open coordination site in any of the substrate-bound structures, a position trans to His180. To obtain experimental evidence that this site is in fact the oxygen binding site, NO was used as an O2 mimic. Similar to previously observed structures of S-HPP Fe(II) HppE,15 the structure of S-HPP Fe(II) HppE NO at 2.85 Å resolution reveals bidentate coordination of the substrate to the Fe(II) center via the C2 hydroxyl and the phosphonate oxygen in all three molecules in the asymmetric unit. In one of the three monomers, there is density only for a water molecule and not for NO (Supporting Information Figure 1h). However, in the other two monomers (monomers A,B (Fe S-HPP NO)), NO is coordinated to the Fe(II) center as seen by omit and positive difference electron density that is consistent with a diatomic molecule (Figure 4a,b; also see Supporting Information Figure 1f,g). When a water molecule is refined into the electron density of monomers A and B instead of NO, positive difference electron density results, indicating the presence of a second atom (Figure 4b). When NO is refined in this position, no positive or negative difference electron density appears, indicating that a diatomic molecule is the correct size. Because the only diﬀerence between the S-HPP Fe(II) NO structure and the previous S-HPP bound Fe HppE structures is the soaking of the crystals with NO, we have modeled and reﬁned NO in this site in these two monomers. We ﬁnd that, as predicted, NO coordinates trans to the His180 iron ligand, presumably via the nitrogen atom in the site that was “open” in the substrate-bound structure. The Fe N bond distances in the two NO bound monomers are 1.64 and 1.66 Å, and the Fe N O bond angles are 160 and 161 , which are typical values for iron nitrosyl interactions in model complexes.29 The oxygen atom of the NO is pointed toward C1 of the substrate at an average distance of 3.5 Å. C1 is the proposed carbon from which a hydrogen atom is abstracted.9 While it was predicted previously that Lys23 could interact with the active oxygen 11265 dx.doi.org/10.1021/ja2025728 |J. Am. Chem. Soc. 2011, 133, 11262–11269