Domain Swapping in Inducible Nitric-oxide Synthase (original) (raw)
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Journal of the American Chemical Society, 2009
The NOS ouput state for NO production is a complex of the FMN-binding domain and heme domain, and thereby it faciltates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN/heme interactions are important in formation of the output state by guiding the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bi-domain oxygnease/FMN construct have been observed by using low-temperature MCD spectroscopy. The comparative MCD study of wild type and mutant proteins clearly indicate that a properly docked FMN domain contributes to the observed L-Arg-perturbation of heme MCD spectrum in the wild type protein, and that the conserved surface residues at the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.
Journal of Biological Chemistry, 1996
prised of an oxygenase domain containing heme, tetrahydrobiopterin, the substrate binding site, and a reductase domain containing FAD, FMN, calmodulin, and the NADPH binding site. Enzyme activity requires a dimeric interaction between two oxygenase domains with the reductase domains attached as monomeric extensions. To understand how dimerization activates iNOS, we synthesized an iNOS heterodimer comprised of one fulllength subunit and one histidine-tagged subunit that was missing its reductase domain. The heterodimer was purified using nickel-Sepharose and 2,5-ADP affinity chromatography. The heterodimer catalyzed NADPHdependent NO synthesis from L-arginine at a rate of 52 ؎ 6 nmol of NO/min/nmol of heme, which is half the rate of purified iNOS homodimer. Heterodimer NO synthesis was associated with reduction of only half of its heme iron by NADPH, in contrast with near complete heme iron reduction in an iNOS homodimer. Full-length iNOS monomer preparations could not synthesize NO nor catalyze NADPH-dependent heme iron reduction. Thus, dimerization activates NO synthesis by enabling electrons to transfer between the reductase and oxygenase domains. Although a single reductase domain can reduce only one of two hemes in a dimer, this supports NO synthesis from L-arginine.
Journal of Biological Chemistry, 2006
3 The abbreviations used are: NOS, nitric-oxide synthase; eNOS, endothelial NOS (NOSIII); iNOS, inducible NOS (NOSII); nNOS, neuronal NOS (NOSI); cNOS, constitutively expressed NOS (eNOS and nNOS); CaM, calmodulin; oxyFMN, oxygenase domain/ FMN binding domain construct, as defined in Fig. 2; H 4 B, (6R,6S)-2-amino-4-hydroxy-6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropteridine (tetrahydrobiopterin); EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; L-NOHA, N-hydroxy-L-arginine; epr, electron paramagnetic resonance; Bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3diol; sq, semiquinone.
N-terminal domain swapping and metal ion binding in nitric oxide synthase dimerization
The EMBO Journal, 1999
Nitric oxide synthase oxygenase domains (NOS ox ) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOS ox reveal that conformational changes in a switch region (residues 103-111) preceding a pterin-binding segment exchange N-terminal β-hairpin hooks between subunits of the dimer. N-terminal hooks interact primarily with their own subunits in the 'unswapped' structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N-terminal hooks rearrange from intra-to intersubunit interactions in the 'swapped structure', and Cys109 forms a self-symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N-terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOS ox molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three-dimensional domain swapping of the N-terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.
Proceedings of the National Academy of Sciences, 1996
For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) wer...
Control of Nitric Oxide Synthase Dimer Assembly by a Heme-NO-Dependent Mechanism
Homodimer formation is a key step that follows heme incorporation during assembly of an active inducible nitric oxide synthase (iNOS). In cells, heme incorporation into iNOS becomes limited due to interaction between self-generated NO and cellular heme [Albakri, Q., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 5414-5421]. Here we investigated if NO can regulate at points downstream in the process by inhibiting dimerization of heme-containing iNOS monomer. Heme-containing monomers were generated by treating iNOS dimer or iNOS oxygenase domain dimer (iNOSoxy) with urea. Both monomers dimerized when incubated with Arg and 6R-tetrahydrobiopterin (H 4 B), as shown previously [Abu-Soud,
Heme Distortion Modulated by Ligand-Protein Interactions in Inducible Nitric-oxide Synthase
Journal of Biological Chemistry, 2004
The catalytic center of nitric-oxide synthase (NOS) consists of a thiolate-coordinated heme macrocycle, a tetrahydrobiopterin (H4B) cofactor, and an L-arginine (L-Arg)/N-hydroxyarginine substrate binding site. To determine how the interplay between the cofactor, the substrates, and the protein matrix housing the heme regulates the enzymatic activity of NOS, the CO-, NO-, and CN ؊ -bound adducts of the oxygenase domain of the inducible isoform of NOS (iNOS oxy ) were examined with resonance Raman spectroscopy. The Raman data of the CO-bound ferrous protein demonstrated that the presence of L-Arg causes the Fe-C-O moiety to adopt a bent structure because of an H-bonding interaction whereas H4B binding exerts no effect. Similar behavior was found in the CN ؊ -bound ferric protein and in the nitric oxide (NO)-bound ferrous protein. In contrast, in the NO-bound ferric complexes, the addition of L-Arg alone does not affect the structural properties of the Fe-N-O moiety, but H4B binding forces it to adopt a bent structure, which is further enhanced by the subsequent addition of L-Arg. The differential interactions between the various heme ligands and the protein matrix in response to L-Arg and/or H4B binding is coupled to heme distortions, as reflected by the development of a variety of out-of-plane heme modes in the low frequency Raman spectra. The extent and symmetry of heme deformation modulated by ligand, substrate, and cofactor binding may provide important control over the catalytic and autoinhibitory properties of the enzyme.
Journal of Biological Chemistry, 2004
about 20-fold slower than in mammalian NOSs. Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile. To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iN-OSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics. The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes. Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions. I224V bsNOS displayed three heme transitions involving four species as typically occurs in wildtype NOS, the beginning ferrous enzyme, a ferrous-dioxy (Fe II O 2 ) intermediate, Fe III NO, and an ending ferric enzyme. The rate of each transition was increased relative to wild-type bsNOS, with Fe III NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe II O 2 , a mixture of Fe III NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iN-OSoxy, with the Fe III NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.