Structural Basis of Biological N2O Generation by Bacterial Nitric Oxide Reductase

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Science  17 Dec 2010:
Vol. 330, Issue 6011, pp. 1666-1670
DOI: 10.1126/science.1195591


Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N2O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (FeB) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.

Nitrous oxide gas (N2O) is now the greatest threat to the ozone layer and also induces climate change as a greenhouse gas more powerful than carbon dioxide and methane (1). Agricultural fertilizers, fossil fuel combustion, biomass burning, and animal waste contribute to N2O production. However, the largest emission source of N2O into the atmosphere is bacterial breakdown of nitrogen compounds in soils and in the oceans. Denitrifiers perform the step-by-step chemical reduction of nitrogen oxides (NO3 and NO2) to N2, producing N2O as an intermediate by-product: NO3 → NO2 → NO → N2O → N2. The key enzyme in N2O production is nitric oxide reductase (NOR), which catalyzes the reduction of nitric oxide (NO) with two electrons and two protons: 2NO + 2e + 2H+ → N2O + H2O. The NOR reaction is also of interest to synthetic chemists because it involves N-O bond cleavage and N-N bond formation (Scheme 1). Structural and functional models of the active site of NOR have been synthesized (2), and recently a construction of the active site of NOR in myoglobin through biomolecular engineering techniques was reported (3, 4). In addition, NOR is clinically and pharmaceutically important as well because pathogens such as Pseudomonas aeruginosa, which are known to be a major opportunistic pathogen that causes acute and chronic infection, have NOR to detoxify cytotoxic NO produced in anaerobic respiration in cystic fibrosis lung (5). Accordingly, the molecular mechanism of the NO reduction by NOR have been extensively studied through chemical, biochemical, and physicochemical techniques (2, 68), but a structure of NOR enzyme has been lacking.

Scheme 1

Here, we report the structure of cNOR, which has a cytochrome c subunit as the electron donor for the catalytic reaction at the active center. The enzyme was isolated from P. aeruginosa and crystallized in the presence of the cNOR antibody (Fab), which was essential for obtaining high-quality crystals (table S1) (9). Compared with the amino acid sequences of cNOR from Paracoccus denitrificans and Pseudomonas stutzeri, both of which have been comprehensively studied (6, 7), the amino acid sequence of P. aeruginosa cNOR has 75 and 94% homology, respectively (fig. S1). P. aeruginosa cNOR also has five well-conserved Glu residues (Glu135, Glu138, Glu211, Glu215, and Glu280) that have been shown to be functionally important (1012). In addition, the NO reduction activity (433 ± 70 μM NO/min/μM enzyme) is comparable, and the spectroscopic properties are also identical among the cNORs (13, 14).

P. aeruginosa cNOR is an integral membrane and iron-containing heterodimeric enzyme consisting of small (NorC) and large (NorB) subunits (Fig. 1). The NorB subunit has 12 α-helices and interacts with one α-helix of the NorC subunit. These 13 helices span the transmembrane (TM) region. The TM domain of the NorB subunit contains two heme irons in hemes b and b3 and one non-heme iron, FeB (Fig. 1B). As predicted (15), three His residues (His60, His347, and His349) are ligands for the two b-type hemes. The globular hydrophilic domain of the NorC subunit contains heme c, which has His65 and Met112 as axial iron ligands. Consistent with the previous prediction that NOR is an evolutionary progenitor of cytochrome oxidases, the topology of the TM region and the arrangement of the metal centers in cNOR are similar to those of cytochrome oxidases, a superfamily of enzymes that act as the terminal oxidases in aerobic respiratory transport chains (Fig. 1C and fig. S2) (15, 16).

Fig. 1

(A) Overall structures of cNOR from P. aeruginosa viewed parallel to the membrane. The NorB and NorC subunits are shown as ribbon in various colors and white, respectively. Heme c is shown as blue sticks, and hemes b and b3 are shown as red sticks. FeB and calcium ions are represented as orange and green spheres, respectively. (B) Arrangement of metal centers of cNOR. Distances between redox centers are shown. (C) Arrangement of membrane-spanning helices of cNOR viewed from the periplasmic side. Transmembrane helices are indicated with Roman numerals. (D) Cytochrome ba3 oxidase from T. thermophilus (PDB code 1EHK). Subunit 1 with helices I ~ XII is shown in color. Helix I of subunit 2 and helix I of subunit 3 are shown in gray. The position of the K-pathway is marked with an asterisk.

Non-heme iron FeB and heme b3 constitute the binuclear center that serves as the catalytic site of cNOR (Fig. 2A and fig. S3). For this analysis, cNOR was crystallized in a purified ferric resting form (fig. S3) (8, 17). Three His residues (His207, His258, and His259) coordinate the non-heme FeB, and Glu211 also serves as a ligand, as was proposed for the homologous Glu198 in P. denitrificans cNOR (4, 10, 18). The FeB site has a slightly distorted trigonal-bipyramidal coordination geometry that differs from the geometry observed at the CuB site in cytochrome oxidase (Fig. 2). The Glu211 coordination position of FeB in cNOR is occupied by a His ligand (His233) of CuB in cytochrome oxidases; this His ligand is covalently attached to a highly conserved Tyr residue (Tyr237 of Thermus thermophilus cytochrome oxidase) (fig. S4B) (1921). The carboxylate of the coordinated Glu211 is hydrogen-bonded to Glu280. Glu215 and Glu280, both of which are also conserved Glu residues in NORs, are near the binuclear center, indicating a large electro-negative environment at the active site, as predicted on the basis of resonance Raman studies (8). This electro-negative environment results in a redox potential of heme b3 iron (60 mV) that is lower than those of hemes b (345 mV) and c (310 mV) irons and thus contributes to activation of the NO molecule bound to the heme b3 and FeB irons (3, 4). In addition, the hydrogen-bonding network involving the three Glu residues can serve as a terminal proton-donating system to facilitate N-O bond cleavage in N2O and H2O production (10, 12).

Fig. 2

(A) Structure of the binuclear center of cNOR. Non-heme iron (FeB) is coordinated by Glu211, His207, His258, and His259 that are conserved in cNOR. Glu211 is involved in hydrogen bond network with two other conserved acidic residues, Glu215 and Glu280. The proximal His347 of heme b3 is hydrogen-bonded to the carbonyl group of Gly321, as observed in cytochrome ba3 or aa3 oxidase. In contrast, Glu side chain interacts with proximal His of cbb3 oxidase, which would increase the reactivity of the bound O2. Unlike the O2 complex of oxidase, NO complex of cNOR would form the penta-coordinated ferrous heme b3 (21). In such a case, highly anionic proximal His is not preferred, which is consistent with the present structure. (B) Structure of the binuclear center of ba3 oxidase (PDB code 1XME). His233 and Tyr237 are connected by a covalent bond. (C) Coordination structure of calcium and the interaction of the heme propionates. An anomalous difference Fourier map calculated from the low-energy data set collected at wavelength of 1.9 Å is contoured at 5 σ and shown as blue mesh. One of the calcium ligands (Glu135) and the residues interacting with the D-ring propionate of heme b3 (Asn335, His339, Gly340, and Thr344) are conserved in the amino acid sequence of other cNOR and cbb3 oxidase, whereas cbb3 oxidase structure lacks the interaction with Gly or Thr.

Three possible mechanisms have been proposed for the reduction of NO by NOR: the trans mechanism (8, 14, 22), the cis FeB mechanism (23, 24), and the cis heme b3 mechanism (25, 26). Because two NO molecules are required for NOR reaction, the heme b3 and FeB binuclear pocket allows two NO molecules to bind in all three proposed mechanisms. This is in sharp contrast with the heme a3 and CuB binuclear center of cytochrome oxidases, which can accommodate a single O2 molecule. Slightly larger space near the catalytic binuclear center for substrate-binding is created by the residues of FeB ligand residues and hydrophobic residues in cNOR as compared with cytochrome oxidases (fig. S4), although the size of the space in cNOR is not opened enough to accommodate two NO molecules. This is caused by the short distance between the two irons of cNOR (3.9 Å) in comparison with the distance between the heme a3 iron and CuB in cytochrome oxidase (4.4 Å) and by the ligation of the Glu211 to FeB. When the iron is reduced from the ferric state to the ferrous state, the μ-oxo ligand dissociates, and the distance between the two irons probably increases. Upon NO binding to reduced iron during the catalytic cycle of cNOR, further conformational changes of the binuclear center and its protein ligands might be required to locate the two NO molecules suitable for N-N bond formation. To study this issue, we need to determine structures of cNOR in several oxidation and ligand-coordination states.

The electrons used in the NO reduction are donated from cytochrome c551 to the heme c, which is covalently attached at Cys61 and Cys64 in the globular domain of the NorC subunit (fig. S5). The binding site of cytochrome c551 probably overlaps Fab binding site on cNOR (fig. S6) because Fab can inhibit cytochrome c551-supported NO reduction but not phenazine methosulfate (PMS)–supported NO reduction (fig. S7 and table S2). Electrons thus donated are transferred from the heme c iron to the heme b iron (20.5 Å distance) through the NorC/NorB interface (fig. S8). The iron-to-iron distance between the hemes b and b3 in cNOR is 14.1 Å, which is comparable with that between hemes a and a3 in cytochrome oxidase, and Gly348 is present between the two hemes in cNOR (Fig. 1B) in place of conserved Phe on cytochrome oxidases (19). A calcium ion (Ca) was identified in cNOR (Fig. 2C, fig. S2, and table S3) at the same position of micro-aerobic respiratory enzyme, cbb3 oxidase (27). Because the plausible electron transfer pathway in cNOR (figs. S8 and S9) from the heme c to the binuclear center (heme b3 and FeB) appears similar to the pathway from CuA to heme a in cytochrome oxidases (19, 21, 28), Ca possibly acts as a factor maintaining protein and heme structures suitable for the intramolecular electron transfer.

In the proposed mechanism of NO reduction (8, 14, 2226), a hyponitrite (ONNO) is plausibly formed as a transient species after the N-N bond formation between the two bound NO molecules (Scheme 1). To facilitate N-O bond cleavage of the transient species to produce N2O and H2O, protons must be transferred from bulk water to the buried active site, possibly through a water channel and a hydrogen-bonding network. We identified two channels extending from the NorB/NorC interface to the periplasmic side of the enzyme (Fig. 3B), which connect the bulk water and the catalytic center of the enzyme through a possible hydrogen-bonding network (Fig. 3C). Although a long gap between Thr330 and the propionate of heme b3 (8.0 Å) is observed in the channels and hydrogen-bonding network, introduction of one or two water molecules in the gap would facilitate proton transfer to the binuclear center through Glu211, Glu215, and Glu280. A branched hydrophobic channel extending from the binuclear center to the surface in the membrane-spanning region that could provide a means of NO entry was identified (Fig. 3, orange), but neither an obvious channel nor a hydrogen-bonding network from the active site of the cNOR to the cytoplasmic region was identified (Fig. 4), which is in sharp contrast to the K- and the D-channel of cytochrome oxidases, which are considered as possible proton transfer channels (1921).

Fig. 3

(A) The hydrophobic channel (orange surface) connecting the binuclear center and membrane lipid surface is divided into two pathways. Only the NorB subunit is shown as ribbon. (B) Two hydrophilic channels (magenta and green surface) at the interface between NorC and NorB subunits connect the periplasmic space and calcium site. The NorC and NorB subunits are shown as blue and white ribbon, respectively. (C) Possible pathways for delivering protons and water. Waters observed in the crystal structure are shown as yellow spheres. Residues from NorC and NorB subunits are shown as blue and green stick models. The first hydrophilic channel in the subunit interface, represented by a magenta surface in panel (B), starts from Glu57 and ends at the propionate of heme b3. Seven waters are observed in this channel and generate the hydrogen-bonding network involving the side chains of the residues (Lys53 and Glu57 of NorC and Glu135 and Asp198 of NorB) that are highly conserved in cNOR. The second hydrophilic channel between Gly340 and the heme c propionate, shown as a green surface in (B), contains nine waters that interact with the main chain carbonyl group or the side chain of the conserved residues. This hydrogen-bonding network is separated by 8.0 Å from another network involving the FeB ligand Glu211.

Fig. 4

Residues of cNOR corresponding to the (A) K-pathway and (B) D-pathway of cytochrome aa3 oxidase are mostly hydrophobic. The main chain of NorC and NorB subunits is shown as blue and green ribbons, respectively. Comparisons with the residues of cbb3, aa3, or ba3 oxidase in the same region are shown in fig. S11.

In 1985, the first data showing that the protons required for NO reduction are supplied from the periplasmic space were obtained from whole-cell measurements (29). Membrane potential measurements of Rhodobacter capsulatus by using endogenous carotenoid pigments indicated that the NOR reaction is not electrogenic (30). Since then, extensive evidence including electrochemical, biochemical, and flow-flash kinetic data have been obtained, all of which unambiguously indicate that electrons and protons are supplied from the periplasmic space. Therefore, we propose that the channels and hydrogen-bonding network identified in the P. aeruginosa cNOR structure serve as the pathway for proton transfer in the catalytic reduction of NO at the binuclear center. Asp198 (NorB) and Glu57 (NorC) at the protein surface are probably the proton entry site, because both acidic residues are highly conserved in NORs (fig. S1).

Glu135 in P. aeruginosa cNOR, which is one of the conserved Glu residues (fig. S10), participates in the formation of the proposed proton transfer network (Fig. 3C). This is consistent with the proposal by Reimann et al. for the corresponding Glu122 in P. denitrificans cNOR (31). Glu135 also serves as a Ca ligand (Fig. 2C), suggesting that it is a key residue in determining the structural and functional properties of cNOR. Indeed, Flock et al. reported that upon mutation of Glu122 in P. denitrificans cNOR, the NO reduction activity was significantly decreased (11). This mutation effect might be caused by Ca dissociation, disturbance of electron transfer between the hemes, and disturbance of proton transfer to the binuclear center.

Similarity in the overall structure, the hydrogen-bond network from heme b3 propionate to periplasmic space, metal configuration, and Ca ligation between cNOR and cbb3 oxidase suggests that two enzymes are very close in the phylogenetic tree, although it does not necessarily support the notion that aerobic respiration evolved from nitrate respiration. Structural differences of the binuclear center, however, provide insight into conversion of the chemical reactivity of the enzymes from the NO reduction to the O2 reduction. The Glu211 in cNOR is a better ligand for Fe, whereas the His-Tyr covalent bond in cytochrome oxidases would restrain the geometry of non-heme metal to a more planar form that is favorable for Cu atom coordination. Thus, the amino acid substitution determines the selectivity of non-heme metal, which could lead to the conversion of respiratory substrate. This functional conversion of respiratory enzyme must have played a key role in the evolution of life to adjust to the environmental change of earth.

Considering the regions of cNOR that correspond to the K- and D-proton–pumping pathways proposed for cytochrome oxidases (Fig. 4), there are many hydrophobic residues, but neither an obvious channel nor a hydrogen-bonding network can be identified (comparison with cytochrome oxidases is shown in fig. S11), indicating no connection between the cytoplasm and periplasm (32). This observation is consistent with the absence of proton-pumping ability of cNOR. However, near the terminal proton donor Glu211, cNOR contains a small charged region generated by Glu215, Glu280, and water (Fig. 4A) that partly overlaps with the K-pathway of cytochrome oxidases. This region of cNOR might reflect the first step in the evolution to acquire a proton delivery pathway from cytoplasm to the binuclear center. Indeed, in the structure of cbb3 oxidase (27) the proton permeable route between the active site and cytoplasm is found at the region equivalent to the K-pathway, whereas the putative D-pathway is blocked by hydrophobic residues. Further structural, biochemical, and chemical studies that are based on the structural characterization of cNOR and cbb3 oxidase are expected to elucidate the evolutionary history of the heme copper oxidase superfamily.

Supporting Online Material

Materials and Methods

Figs. S1 to S11

Tables S1 toS3


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Atomic coordinates and structure factors have been deposited to the Protein Data Bank (PDB) with code 3O0R. We thank T. Tosha for measurements of the enzymatic activity of cNOR under various conditions and the staff of the SPring-8 beamlines for their help with diffraction measurements. S.N., H.S., and Y.S. are supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and Y.S. also acknowledges financial support from the Structural Biology and Molecular Ensemble projects of RIKEN. A part of this work was also supported by a grant from the ERATO IWATA Human Receptor Crystallography Project from the Japan Science and Technology Agency.
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