Antonyuk et al. 10.1073/pnas.0504207102. |
Fig. 5. Thermal ellipsoids (50%) of the type 2 Cu site and proton delivery channel for the resting-state enzyme (a), the nitrite-soaked enzyme (b), and the endogenously bound NO enzyme (c).
Fig. 6. The overall charge distributions on the accessible surfaces of the resting-state structures of Achromobacter cycloclastes nitrite reductase (AcNiR) (a) and Alcaligenes xylosoxidans NiR (AxNiR) (b) (PDB ID code 1OE1). These data were calculated by using GRASP (38) for 0.9- and 1.04-Å resolution coordinates, respectively. The orientation of each trimer molecule has been chosen to highlight the position of the substrate entry channel (marked by "X"). The potential surface of AcNiR is strongly negative (net electron charge is –27e), whereas the accessible surface charge of AxNiR (net charge –6e) is largely neutral with some areas of negative (red) and positive (blue) charge. In both enzymes, the surface at the site of substrate entry is negatively charged.
Table 2. Crystallographic data collection, processing, and refinement statistics (additional information)
| Resting enzyme | Nitrite-soaked | Endogenous NO | Endogenous nitrite plus NO |
I /s(I) last shell | 2.0 | 2.0 | 2.1 | 2.0 |
ML B factor ESU, Å2 | 0.40 | 0.84 | 0.99 | 0.98 |
RMS bonds, Å | 0.018 | 0.018 | 0.021 | 0.020 |
Supporting Text
Supporting Experimental Procedures
Growth of the Organism and Protein Purification.
Achromobacter cycloclastes strain 1013 was obtained from the IAM culture collection (University of Tokyo, Tokyo). For the studies reported here, cultures were grown under denitrifying conditions in a nutrient broth/yeast extract/glycerol medium containing 0.5 g/liter KNO3 (1), modified by the inclusion of 5 mM CuSO4 (2). Cultures were grown anaerobically at 30°C for 50 h in a 200-liter fermenter stirred at 50 rpm. To minimize the accumulation of toxic levels of nitrite, three separate additions of 0.5 g/liter KNO3 and 5 ml of glycerol were added at ~12-h intervals. Cells were harvested by using a continuous centrifuge and frozen in liquid nitrogen before storage at –80°C.Crude extracts were prepared by resuspension of the frozen cells in 50 mM phosphate buffer (pH 7; 2 ml/g) followed by disruption using two passages through a Manton-Gaulin homogenizer at 600 kg/cm2. A small amount of DNase was added before centrifugation at 15,000 ´ g for 90 min to remove cell debris and unbroken cells. To prevent autoreduction and subsequent inactivation of nitrite reductase (NiR) (3), hydrogen peroxide (10 ml of 30% vol/vol) was added to the resulting crude extract (110 ml), which was then loaded onto a column of DEAE cellulose (5 × 15 cm) equilibrated with 20 mM phosphate buffer (pH 7). The column was washed with 500 ml of 20 mM phosphate buffer to remove unbound proteins and then with 500 ml of 100 mM phosphate buffer. It was then developed with a 2-liter linear gradient of NaCl from 100 to 250 mM in phosphate buffer. Separation of colored proteins on the column was followed visually. Pseudoazurin did not bind under these conditions and was present in the flow-through during loading of the crude extract. The gradient resulted in a predominantly greenish band containing Achromobacter cycloclastes NiR (AcNiR) activity that was partially resolved from a purplish band containing nitrous oxide reductase (N2OR) activity.
Ac
NiR used in this study was purified from the N2OR fraction by chromatography on hydroxyapatite (Bio-Rad). This fraction from the DEAE column was loaded onto a 2.5 × 5.5-cm column of hydroxyapatite equilibrated with 100 mM Tris·HCl buffer (pH 7.4) and after washing with 50 ml of loading buffer the column was developed stepwise with buffer containing increasing concentrations of phosphate (50 ml of 5 mM; 30 ml of 10 mM; 40 ml of 20 mM; and 40 ml of 50 mM). A green band of NiR eluted between 10 and 20 mM phosphate and a purple band of N2OR at 20-50 mM phosphate. Further purification of NiR to homogeneity, as judged by SDS/PAGE, was achieved either by gel filtration on S-200 in 50 mM Tris·HCl buffer (pH 8.0) containing 200 mM NaCl or by bulk crystallization. A solution of 4 M ammonium sulfate in 50 mM acetate buffer (pH 4.75) was added to the NiR fraction to a final concentration of 2.0 M. The precipitated proteins were separated from the supernatant by centrifugation for 5 min at 16,000 ´ g. Several AcNiR crystals were added to the supernatant, resulting in bulk crystallization of AcNiR. These crystals were collected by centrifugation and washed in 50 mM acetate buffer (pH 4.75). Crystals were redissolved in 50 mM Mes buffer (pH 6.5). This solution was then used for vapor diffusion crystallization.Supporting Results and Discussion
The Resting-State Active-Site Water Network.
Water molecule W4 (Fig. 1) has been modeled with two positions separated by 1.8 Å, an arrangement that allows a H-bond (2.8–3.0 Å) to be maintained with both conformations of the Asp-98 side chain. A solvent molecule, W5, linked to Ala-137 via an H-bond to the carbonyl oxygen is also modeled with two positions, 1.4 Å apart, one of which is H-bonded to W4 at 2.8 Å. These water molecules were all modeled with partial occupancies. W2 was modeled with 20% occupancy in a position close to the Asp-98 side chain. When Asp-98 is in the gatekeeper conformation, W2 forms a weak H-bond with it at 3.3 Å. When Asp-98 is in its main proximal conformation, toward the proton channel, W2 is excluded by the side chain atoms (see the NO-bound structure below; Fig. 3). W2 is also H-bonded (2.7 Å) to the O atom of Leu-106 when this residue adopts its secondary (lower occupancy) conformation. When Leu-106 is in its primary orientation, corresponding to the proximal conformation of Asp-98, the O atom of Leu-106 is instead H-bonded to W4 at 2.8 Å. Thus, a network of water molecules, extending from the active site to the surface of the enzyme, link the catalytically important Asp-98 residue with residues Leu-106 and Ala-137, which are part of the substrate entry pocket. This network is completed by H-bonding between the Cu-ligating W1 and W3 molecules (2.6 Å) and between W3 and W4 (their closest approach is 3 Å).These features of the water network are conserved in the structures of the substrate/product bound adducts.
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