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Structure of Nitric Oxide Synthase Oxygenase Dimer with Pterin and Substrate

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Science  27 Mar 1998:
Vol. 279, Issue 5359, pp. 2121-2126
DOI: 10.1126/science.279.5359.2121

Abstract

Crystal structures of the murine cytokine-inducible nitric oxide synthase oxygenase dimer with active-center water molecules, the substrate l-arginine (l-Arg), or product analog thiocitrulline reveal how dimerization, cofactor tetrahydrobiopterin, and l-Arg binding complete the catalytic center for synthesis of the essential biological signal and cytotoxin nitric oxide. Pterin binding refolds the central interface region, recruits new structural elements, creates a 30 angstrom deep active-center channel, and causes a 35° helical tilt to expose a heme edge and the adjacent residue tryptophan-366 for likely reductase domain interactions and caveolin inhibition. Heme propionate interactions with pterin and l-Arg suggest that pterin has electronic influences on heme-bound oxygen. l-Arginine binds to glutamic acid–371 and stacks with heme in an otherwise hydrophobic pocket to aid activation of heme-bound oxygen by direct proton donation and thereby differentiate the two chemical steps of nitric oxide synthesis.

Nitric oxide synthases (NOSs) oxidize l-Arg to synthesize nitric oxide (NO), which is a key intercellular signal and defensive cytotoxin in the nervous, muscular, cardiovascular, and immune systems (1, 2). Neuronal NOS (nNOS, NOS1) and endothelial NOS (eNOS, NOS3) produce low NO concentrations for neurotransmission, insulin release, penile erection, vasorelaxation, oxygen detection, and memory storage, whereas cytokine-inducible NOS (iNOS, NOS2) produces larger NO concentrations to counter pathogens and coordinate the T cell response (1). NOSs catalyze two sequential, mechanistically distinct, heme-based oxidations in the five-electron oxidation of l-Arg tol-citrulline (l-Cit) and NO. First,l-Arg is hydroxylated to Nω-hydroxy-l-arginine (NOH–l-Arg) by a mixed-function oxidation (2) analogous to reactions catalyzed by the cytochrome P-450s with a proposed oxo-iron intermediate [P·-Fe(IV)=O, where P· is a porphyrin π-cation radical] (3). Second, NOH–l-Arg is converted tol-Cit and NO by an unusual mechanism involving a one-electron oxidation of NOH–l-Arg and a proposed peroxo-iron intermediate [P-Fe(III)-OO2−] (2).

Each NOS isozyme contains a catalytic NH2-terminal oxygenase domain (NOSox, residues 1 to 498 for iNOS) and a COOH-terminal electron-supplying reductase domain (NOSred, residues 531 to 1144 for iNOS) (2). NOSox binds heme (iron protoporphyrin IX), tetrahydrobiopterin [(6R,1′R,2′S)-5,6,7,8 tetrahydrobiopterin or H4B], and substrate l-Arg. NOSred is homologous to cytochrome P-450 reductase and binds flavin mononucleotide, flavin adenine dinucleotide, and the reduced form of nicotinamide adenine dinucleotide phosphate. An intervening calmodulin-binding region (residues 499 to 530 for iNOS) regulates reduction of NOSox by NOSred. In all three isozymes, H4B binding and NOSoxdimerization are essential for catalytic activity (2). Recently, structures of monomeric murine iNOSox Δ114 (residues 115 to 498) revealed the unusual topology and heme environment of NOSox (4) but lacked H4B and NH2-terminal residues 77 to 114, components required for activity.

Here we report three structures of fully functional, pterin-loaded, dimeric murine macrophage iNOSox [residues 66 to 498 (5)]: with substrate l-Arg (H4B-ARG, 2.6 Å resolution), with product analog thiocitrulline (SCit) (H4B-SCIT, 2.7 Å resolution), and without bound ligands (H4B-H2O, 2.6 Å resolution) (6-8). In the symmetric iNOSoxdimer (90 by 70 by 45 Å3, Fig.1), mobile and exposed hydrophobic regions identified in the iNOSox Δ114 monomer (4) refold to buttress the substrate-binding channel and sequester two molecules of H4B within two symmetry-related helical lariats (Figs. 1 and 2A) at the heart of the extensive dimer interface (2800 Å2 of buried surface area from more than 85 residues per subunit, Fig. 2B). Except for these drastic changes at the interface (Fig. 2C), each subunit maintains the monomer's unusual winged β-sheet fold that resembles a left-handed baseball catcher's mitt (4) (Fig. 1). The two helical lariats (Figs. 1 and 2A), each composed of two 310helices, α10 (residues 454 to 459) and α11 (residues 463 to 471, kinked at residue 467 into α11a and α11b), associate around the two pterins, each of which bridges (Fig. 3A) the COOH-terminal ends of α10 and α11a* (stars indicate the symmetry-related subunit). Helical T's, named for the T shape formed from α8 and α9 (Fig. 2), self-associate symmetrically to frame the helical lariats and bound pterins (Figs. 1B, 2A, and 2B). The NH2-terminal residues not present in iNOSoxΔ114 (primed nomenclature) form an NH2-terminal hook (composed of antiparallel strands β1′ and β2′, residues 77 to 100) that reaches across to the other subunit to interact with β12* and the COOH-terminal end of α9* (Figs. 1 and 2) and thereby influence iNOS dimer formation and activity (5, 9). An irregular extended strand comprising residues 108 to 114 (the NH2-terminal pterin-binding segment) makes important contacts with the pterin (Figs. 2 and 3) and is bridged to the NH2-terminal hook by Glu473*. In the crystal, Cys109, the only isozyme-conserved cysteine to affect dimer formation and H4B binding in iNOS and nNOS (5,10), forms a symmetric disulfide bond across the dimer interface (Fig. 1A). The preceding, disordered, isozyme-variable surface loop (residues 101 to 107) is near its own symmetry mate (Fig. 1B), suggesting that the NH2-terminal hooks may swap from intersubunit to intrasubunit interactions during dimer-to-monomer transitions.

Figure 1

NOSox α-β fold, dimer assembly, and likely interaction surface for NOSred and caveolin. (A) The symmetric iNOSox dimer viewed along the crystallographic twofold axis, showing left (and right) subunits with orange (yellow) winged β sheets and flanking blue (cyan) helices. Ball-and-stick models (white bonds with red oxygen, blue nitrogen, yellow sulfur, and purple iron atoms) highlight active-center hemes (left-most and right-most), interchain disulfide bonds (center, foreground), pterin cofactors (white, left-center and right-center), and substrate l-Arg (green left and magenta right). The NH2-terminal ends contribute β hairpins (center top and bottom) to the dimer interface, and the COOH-termini (lower left and upper right) lie 85 Å apart. Gray loops (residues 101 to 107) are disordered. (B) iNOSox dimer shown rotated 90° about a horizontal axis from (A). Each heme is cupped between the inward-facing palm (webbed β sheet) and thumb (magenta loop in front of left heme and green loop behind right heme) of the “catcher's mitt” subunit fold. (C) Solvent-accessible surface (29) of the iNOSox dimer (one subunit red, one subunit blue) oriented as in (B) and color-coded by residue conservation (paler to more saturated represents less conserved to more conserved) in NOSox sequences of known species and isozymes. The heme (white tubes) is also solvent-exposed on the side (left subunit) opposite the active-center channel (right subunit) and surrounded by a highly conserved hydrophobic surface for NOSred and caveolin binding. (Stereo variations of Figs. through 4 are available athttp://www.scripps.edu/~jat.)

Figure 2

Dimer interface structural elements and conformational changes on iNOSox dimerization. (A) Cα trace of one iNOSox subunit viewed directly into the dimer interface. Protein components contributing residues to the extensive dimer interface are separated into six colored regions: NH2-terminal hook (β1′ and β2′, rose), NH2-terminal pterin-binding segment (yellow), substrate-binding helix α7a (magenta), helical T (α8 and α9, green), helical lariat (α10, α11a and 11b, and β12a, cyan), and other residues (pale purple). Pterin (white bonds with blue nitrogens and red oxygens, center) interacts with heme (yellow bonds with blue nitrogens and red oxygens, center left). (B) The buried surface of the dimer interface color-coded by residues contributed from the regions categorized and colored in (A). (C) Superposition of the iNOSox Δ114 monomer (thin ribbons) onto one subunit of the iNOSox Δ65 dimer (wide ribbons). Although the core of the winged β-sheet domain is structurally conserved between the monomer (purple) and the dimer (orange) with a Cα rmsd of 0.8 Å for residues 115 to 336 and 480 to 496, large differences do occur at the dimer interface (foreground), with some residues moving up to 40 Å. Dimerization causes formation of the helical lariat (cyan) from a loop and β strand of the monomer (bottom center, blue and purple), reorganization of the substrate-binding helix (α7a, magenta) and following sequence (background, upper right) from α7 of the monomer, and a large rotation of α9 to form the helical T (green). Heme pyrrole ring A propionate extends from its bent orientation in the monomer (purple, center, front) to interact with the pterin (white, right) in the dimer. The pterin interacts with the NH2-terminal hook (rose), the NH2-terminal pterin-binding segment (yellow), and the helical lariat (cyan).

Figure 3

Pterin interactions in the substrate-binding channel. (A) A σA-weightedF obsF calc omit map (purple 3.5σ and red 9.5σ contours) showing well-ordered H4B (yellow carbon, red oxygen, and blue nitrogen bonds) and its hydrogen bonds (white dashed lines) to each subunit of the dimer (yellow carbons or green carbons). (B) The hydrogen-bond network coupling pterin binding, dimerization,l-Arg binding, and heme activity. Heme (HEM, top right) andl-Arg (ARG, green, top center) are bound by one subunit (magenta Cα trace and yellow side chains, with blue nitrogen and red oxygen balls), whereas H4B (H4B, center edge on) interacts with the helical lariat of the symmetry-related subunit as well (red Cα trace and green side chains, bottom).

Dimerization creates a ∼30 Å deep, funnel-shaped active-center channel from the shallow ∼10 Å distal heme pocket present in the monomeric iNOSox Δ114 structure (4) by refolding and recruiting components of the dimer interface: α7a, the pterin, the NH2-terminal pterin-binding segment, and the NH2-terminal hook (Figs. 1 and 2). Helix α7a (residues 370 to 378, Fig. 2A) supplies residues that interact with both substrate and pterin (Figs. 3 and 4). The channel, which is formed primarily by the residues of one subunit, narrows from an ellipsoid mouth ∼9 by 15 Å2 bracketed by Trp84, Ala276, Gln381, and Glu488 to the l-Arg guanidinium-binding site above the heme's distal face (Figs. 1C and 3B). The zigzag β-strand transitions (4) that structure the winged β sheet may be important for funneling dioxygen to the heme iron as they form the side of the active-center channel that remains open when l-Arg is bound (Figs. 1C and 4A).

Figure 4

Ligand-binding in the active center. (A) The H4B-bound iNOSoxdimer (yellow carbons, red oxygens, and blue nitrogens) with active-center water molecules (red crosses), but without substrates or inhibitors [H4B-H2O (8)], viewed down the active channel and shown with its σA-weighted 2F obsF calc electron density map (contours: purple 1.4σ, red 4.0σ, cyan 10σ). (B) l-Arg in the substrate binding site [H4B-ARG (7, 8)] from a view rotated roughly 180° about a vertical axis compared to (A). The σA-weighted F obsF calc omit electron density map (contours: purple 3.0σ, red 5.5σ), calculated with l-Arg and active-center waters removed from F calc, depicts electron density for l-Arg and its associated water molecule (red crosses, left) in the absence of the minority contribution of the water-bound structure [see (A)] also present in H4B-ARG (8). (C) SCit in the substrate-binding site [H4B-SCIT (7, 8)] viewed as in (B). The sulfur of the SCit thiourea group (green) is directed over the heme iron and highlighted by the highest contour level (cyan) in the σA-weighted F obsF calc SCit omit map (contours: purple 3.5σ, red 8σ, cyan 10σ). The omit electron density maps of (B) and (C) were averaged over noncrystallographic symmetry.

Conformational changes on dimerization expose the heme edge on the side opposite to the active-center channel and provide a likely interaction surface (Fig. 1C) for complementarily shaped NOSred, on the basis of P-450 reductase homology (11). The α9 helix pivots 35° (Fig. 2C) to uncover a hydrophobic pocket, conserved across species and isozymes, that includes Trp366 and the heme pyrrole ring C methyl and vinyl groups (Figs. 1C and 4). In fact, caveolin-1 and -3, which target eNOS to the plasma membrane caveolae and inhibit NO synthesis in a manner reversed by calmodulin (12), bind conserved NOS residues that are included in the surface implicated above for NOSred interactions. Thus, caveolin binding may biologically regulate NOS by blocking NOSred from supplying electrons to the heme via pyrrole ring C (Fig. 3B).

The iNOSox dimer structure supports a fundamental role for pterin H4B in controlling iNOS subunit interactions (13) and active-center formation, but not for pterin directly hydroxylating l-Arg or activating heme-bound oxygen (Fig. 3). H4B stabilizes the dimer by integration into the hydrophobic heart of the interface and facilitates substrate interactions by lining the active-center channel and hydrogen bonding to a heme propionate and to α7, two elements involved inl-Arg binding (Fig. 3). In the NOS dimer, H4B sits proximal and perpendicular to the heme, with its 2-amino-4-hydroxy-pyrimidine ring interacting with the extended propionate from heme pyrrole ring A, its O4- and N5-containing edge presented to solvent in the active-center channel, and its dihydroxy-propyl side chain directed toward the NH2-terminal hook and second subunit (Figs. 1A, 2, and 3). The helical lariats from both subunits sandwich each pterin with aromatic π-stacking interactions, and the NH2-terminal pterin-binding segments provide additional contacts and hydrogen bonds to further stabilize the cofactor sites (Fig. 3).

The pterin is positioned for indirect structural and electronic influences on substrate and inhibitor binding and catalysis (14-17). Hydrogen bonds bridge from pterin N3 (directly) and O4 (through a water molecule) through heme propionate A to thel-Arg α-amino group, and from pterin O4 (directly) and N5 (through a water molecule) to Arg375 of the substrate-binding helix, which participates in an extensive hydrogen-bond network (Fig. 3). In the pterin-free monomer structure (4), heme propionate A interacts with Arg193 and the α7a helix, where it could block substrate binding, consistent with the inability of iNOSox Δ114 to bindl-Arg (5). Pterin-induced changes in the heme environment that include ordering of the active-center channel, increased sequestration of the proximal heme ligand Cys194, and extension of the negative heme A propionate away from the distal heme pocket (Figs. 2C and 3B) may account for the 50-mV increase in heme redox potential (18) and low-to-high spin shift of the ferric heme iron (14, 15) in the presence of H4B. Furthermore, increased basicity of the proximal Cys194 thiolate from sequestration in a more hydrophobic environment (Fig. 4A) may promote oxygen activation as well as the pterin-induced 70-fold increase in autoxidation of the ferrous heme-dioxy complex (19). However, close H4B analogs that promote stable dimerization, substrate interactions, and heme spin shifts yet do not support NO synthesis (14, 20) suggest that the structural coupling between H4B and the heme propionate has a key catalytic role.

Aromatic amino acid hydroxylases (AAHs) use H4B as an electron-supplying cofactor, oxidizing it to 4a-hydroxy-H4B during catalysis (21). The NOS helical lariat contains residues conserved by the AAHs (22) that map near the catalytically important nonheme iron in pterin-free structures of tyrosine and phenylalanine hydroxylases (23). However, NOSs conserve only one of the two iron-binding His residues of AAHs (His471, which contacts the pterin from the symmetry-related subunit) and lack a similar iron site, suggesting the respective pterin-binding sites have adapted for different chemistry. The absence of pterin recycling during steady-state NO synthesis (24) and of redox activity in single-turnover experiments (25) is consistent with H4B modulating oxy-heme reactivity from the heme edge opposite to the NOSredinteraction site without H4B participating in bona fide electron transfer.

In the structure of dimeric iNOSox with H4B but without bound substrate or inhibitor [H4B-H2O (8)] the ferric heme iron is pentacoordinate, and two water molecules interact with each other above the heme's distal face: one hydrogen bonds to substrate-binding residue Glu371 (see below), whereas the other resides 4.2 Å above the heme iron (Fig. 4A). An H4B-mediated change from water-bound hexacoordinate to pentacoordinate heme is consistent with a shift to a majority of high-spin hemes (5, 14, 15) and a 30-fold rate increase in NO binding to the ferric heme iron (26). H4B-promoted dissociation of heme-bound water likely reflects cofactor-induced changes in the heme environment with dimerization and pterin-binding and formation of a more favorable water site above the heme by an extended Glu371 conformation not seen in the NOS monomer.

The substrate l-Arg [H4B-ARG (7,8)] and product analog SCit [H4B-SCIT (8)] bind to the sequence-conserved active center in analogous conformations, suggesting that isozyme-specific inhibitors must interact with more variable, distal channel regions (Fig. 4). Thel-Arg guanidinium and SCit thiourea groups both make two syn hydrogen bonds from their heme-distal and bridging nitrogens to both carboxylate oxygens of Glu371, and one hydrogen bond from their heme-distal nitrogen to the carbonyl of Trp366at the otherwise hydrophobic bottom of the active-center channel (Fig.4, B and C). The NOS porphyrin ring bends significantly to facilitate favorable stacking of the guanidinium or thiourea groups against pyrrole ring A. The Glu371 residue is critical for substrate binding to NOS (27) and mediates inhibitor binding to iNOSox Δ114 (4). Both the l-Arg and SCit carboxylate groups hydrogen bond to the Tyr367hydroxyl, a water molecule bridged to Arg382, and the carboxylate of Asp376, which may be protonated given its hydrogen-bonding partners and replacement by Asn in eNOS (Fig. 4). Interactions of the l-Arg α-amino and carboxylate groups with a network of hydrophilic side chains that are directly linked to structural elements involved in dimer formation (for example, the α7 helix), and to the same heme propionate that ligates H4B (Fig. 3B), likely explain the cooperativity among dimerization, H4B binding, and substrate binding (2, 14, 15,28).

Interactions of the l-Arg guanidinium and SCit thiourea groups at the bottom of the heme pocket (Fig. 4, B and C) suggest a mechanism for NO synthesis where proton donation from substratel-Arg to bound dioxygen facilitates oxo-iron formation for the conversion of l-Arg to NOH–l-Arg, thereby neutralizing the l-Arg guanidinium group and discriminating between oxo- and peroxo-iron species in the two steps of NO synthesis (Fig. 5). It is likely thatl-Arg binds when protonated, given the guanidinium interaction with Glu371 and the heme π-electrons. The analogous conformations of substrate and product analog (Fig. 4, B and C) suggest that the bridging and heme-distal guanidinium nitrogens maintain their hydrogen bonds to Glu371 and Trp366 throughout catalysis. This leaves only the remaining terminal nitrogen well positioned (3.8 Å from the heme iron) and free to first donate a proton to peroxo-iron, facilitating oxygen-oxygen bond cleavage and reduction of the guanidinium charge, and to then react with the remaining electrophilic oxo-iron species to become hydroxylated (Fig. 5). Besides its proximity to the site of oxygen activation (Fig. 4B), l-Arg has a p Ka ( Ka is the acid constant) at least 3 to 4 units less than water, the proposed proton donor to dioxy-iron in cytochrome P-450s (3). Further lowering of the l-Arg p Ka by distortion of its planar guanidinium is suggested by the electron density maps. Once l-Arg is converted to NOH–l-Arg, no proton is available to facilitate the breakdown of peroxo-iron, thereby allowing this dioxygen species to react with NOH–l-Arg to form citrulline and NO. Thus, the ligand-bound NOS structures suggest that NOS catalysis selects between two different reductive activations of dioxygen, depending on the protonation state of the substrate.

Figure 5

Proposedl-Arg–assisted NOS oxygen activation. First, substratel-Arg (only guanidinium shown) donates a proton to peroxo-iron, facilitating O–O bond cleavage and conversion to a proposed oxo-iron(IV) π-cation radical species, which then rapidly hydroxylates the neutral guanidinium to NOH–l-Arg, possibly through a radical-based mechanism (3).

  • * Present address: Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA.

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