Journal of the American Chemical Society ARTICLE Figure 2. Structures of HppE with R-HPP or S-HPP bound in the active site. (a) Bidentate binding mode for R-HPP in the Co(II) HppE structure. (b) Bidentate binding mode for R-HPP in Fe(II) HppE structure. (c) Bidentate binding mode for S-HPP in the Co(II) HppE structure (1ZZB monomer B). (d) Bidentate binding mode for S-HPP in the Fe(II) HppE structure (1ZZ8 monomer C). Substrate and protein residues are shown as sticks (carbon (gray), oxygen (red), nitrogen (blue), phosphorus (purple)), water molecules as spheres (cyan), Co(II) as a sphere (magenta), and Fe(II) as a sphere (rust). The 2Fo Fc maps (blue mesh) are contoured at 1.0 σ, and the Fo Fc omit-maps (green mesh) of the substrate indicating the position of the phosphorus are contoured at 8.0, 10.0, 9.0, and 8.0 σ, respectively. into the well for additional 2 h. Crystals were then transferred to cryo- protectant buffer solution and flash-cooled in liquid nitrogen. Data Collection and Analysis. Data sets for R-HPP Fe(II) HppE and S-HPP Fe(II) HppE NO were collected at the Stanford Synchrotron Radiation Lightsource and the Advanced Photon Source, respectively. The R-HPP Co(II) HppE data set was collected at the Advanced Light Source. The data were integrated and scaled in DENZO and SCALEPACK,25 respectively (Supporting Information Table 1). Structure Determination. Crystals of S-HPP Fe(II) HppE NO (P42212; a,b = 111.66 Å, c = 152.16 Å) and R-HPP Fe(II) HppE (P42212; a,b = 111.67 Å, c = 152.81 Å), with three molecules in the asymmetric unit, were isomorphous with our previous crystals of S-HPP Fe(II) HppE (P42212; a,b = 111.65 Å, c = 152.07 Å). Thus, these structures were solved by rigid body refinement in CNS26 using protein atoms from S-HPP Fe(II) HppE structure15 as an initial model (PDB code 1ZZ8). Model building was done in COOT,27 and iterative refinement steps were accomplished using CNS. Topology and parameter files for R-HPP were obtained using HIC-Up.28 Parameter and topology files for the iron-NO coordination sphere were developed using the bond distances and angles from high-resolution X-ray struc- tures of nonheme iron model complexes with NO bound to iron.29 To allow movement of atoms away from the average values, weak restraints were used. The CNS protocol included simulated annealing against a maximum likelihood function target, positional refinement, and B-factor refinement; noncrystallographic symmetry restraints and sigma cutoff were not used. Because the R-HPP Co(II) HppE crystal, consisting of two mol- ecules in the asymmetric unit (P6522; a,b = 86.30 Å, c = 219.1 Å) was isomorphous with our previous crystals of Tris Co(II) HppE (P6522; a,b = 86.36 Å, c = 220.17 Å), the R-HPP Co(II) HppE structure was solved by rigid body reﬁnement in CNS using only protein atoms from the reﬁned Tris Co(II) HppE structure15 as an initial model (PDB code 1ZZC). Iterative model reﬁnement was performed in CNS using Figure 3. Comparison of the cantilever hairpins (β-strands labeled 1 and 2) of the R-HPP Fe(II) HppE structure (green) and the S-HPP Fe(II) HppE structure (cyan). Bound substrate and selected residues are shown as sticks, and iron atoms are shown as spheres (rust). the protocol described above. Manual adjustment of the model was performed using XFIT.30 Structures of R-HPP Co(II) HppE, R-HPP Fe(II) HppE, and S-HPP Fe(II) HppE NO were analyzed using 2Fo Fc composite omit maps, and Ramachandran geometries were analyzed with PROCHECK.31,32 The results from PROCHECK indicate that the R-HPP Co(II) HppE structure has 89.4% of the residues in the most favored and 10.6% of the residues in the additionally allowed regions of the Ramachandran plot. For R-HPP Fe(II) HppE structure, the Ramachandran plot indicates that 87.7% of the residues are in the most favored and 12.3% of the residues are in the additionally allowed regions. The Ramachandran plot for S-HPP Fe(II) HppE NO structure indicates that 86.2% of the residues are in the most favored, 13.6% of the residues are in the addi- tionally allowed regions, and 0.2% of the residues in the generously allowed regions. Chain A of the R-HPP Co(II) HppE struc- ture contains residues 5 198, while other chains consist of residues 6 198. 11264 dx.doi.org/10.1021/ja2025728 |J. Am. Chem. Soc. 2011, 133, 11262–11269