Alteration of the Heme Prosthetic Group of Neuronal Nitric-Oxide Synthase during Inactivation by NG-Amino-L-arginine in Vitro and in Vivo (original) (raw)
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Effects of Arginase Isoforms on NO Production by nNOS
Nitric Oxide, 2002
Both arginase isoforms (AI and AII) regulate highlevel NO production by the inducible NOS, but whether the arginase isoforms also regulate low-level NO production by neuronal NOS (nNOS) is not known. In this study, 293 cells that stably overexpress nNOS gene (293nNOS cells) were transfected with rat AI (pEGFP-AI) or AII (pcDNA-AII) plasmids, and nitrite production was measured with or without supplemental L-arginine. Transfection with pEGFP-AI increased AI expression and activity 10-fold and decreased intracellular L-arginine by 50%. Nitrite production was inhibited by >80% when no L-arginine was supplemented but not when 1 mM L-arginine was present. The inhibition was reversed by an arginase inhibitor, N -hydroxy-L-arginine. Transfection with pcDNA-AII increased AII expression and activity but had little effect on nitrite production even if no L-arginine was added. These results suggest that, in 293nNOS cells, AI was more effective in regulating NO production by nNOS, most likely by competing for L-arginine.
Biochemistry, 1997
Nitric oxide synthases (NOSs) are proposed to generate NO and citrulline from L-arginine in two steps: initial N-hydroxylation to generate N ω -hydroxyarginine (NOHA) followed by a three-electron oxidation of the hydroxylated nitrogen to form products. Both steps consume NADPH and may involve heme iron-based activation of O 2 . Studies done under multiple-turnover conditions suggest that 0.5 mol of NADPH is consumed to convert 1 mol of NOHA to products, implying that one electron from NADPH may be sufficient. To test this, we studied NOHA oxidation under single-turnover conditions using neuronal NOS (nNOS), whose heme iron reduction requires bound calmodulin. The heme iron in calmodulinbound nNOS was reduced with excess NADPH under anaerobic conditions, calmodulin was then dissociated from nNOS to prevent subsequent heme iron reduction, NOHA was added, and the reaction initiated by exposure to air. Spectra obtained at each step were consistent with buildup of NOHA-bound ferrous nNOS prior to air exposure. Reactions containing graded amounts of nNOS produced L-citrulline in linear relation (1.2 ( 0.1 mol of citrulline per mole of nNOS). Nitrite and nitrate also accumulated as NO-derived products. Control reactions that contained L-arginine instead of NOHA, no enzyme, or ferric nNOS did not generate products. Thus, supplying a single electron from NADPH to the heme iron permits nNOS to catalyze one full round of citrulline and NO synthesis from NOHA upon exposure to O 2 . These data provide a molecular explanation for the NADPH requirement in the second step of the biosynthetic reaction, implicate ferrous-dioxy nNOS as a critical reactant in that step, and eliminate a number of possible alternative catalytic mechanisms or products.
Journal of Biological Chemistry, 2001
We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation. Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS. Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steadystate NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS ؉ CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.
The C331A mutant of neuronal nitric-oxide synthase is defective in arginine binding
1998
It has been proposed that Cys 99 of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH 4) binding. To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys 331 to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme. C331A-nNOS, as isolated, was catalytically incompetent. Upon prolonged incubation with L-arginine (L-Arg), not only BH 4 binding but also catalytic activity could be restored. In contrast to wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm that shifts immediately upon L-arginine addition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited an absorbance maximum at 420 nm. C331A-nNOS, as isolated, did not bind detectable levels of either [ 3 H]N-nitro-L-arginine or [ 3 H]BH 4 , but [ 3 H]BH 4 binding was reinstated after extended incubation with excess L-arginine. On the other hand, C331A-nNOS and WT-NOS were identical with regard to imidazole binding affinity, CaM binding affinity, and rates of cytochrome c and 2,6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conversion of low to high spin heme after extended incubation with high concentrations of L-arginine (0.1-10 mM). The estimated K d for L-arginine binding to C331A-nNOS was two orders of magnitude greater than WT-nNOS (>100 M versus 2-3 M). Here we propose that Cys 331 plays an important role in stabilizing L-arginine binding to nNOS. Our findings suggest that the primary dysfunction in the C331A mutant of nNOS, as isolated, is disruption of the BH 4-substrate binding interactions as broadcast from this mutated cysteine residue. Prolonged incubation with L-arginine appears to cause remodeling of the mutant protein to a form similar to that of WT-nNOS, allowing for normalized BH 4 binding and nitric oxide synthetic activity.
An Inducible Nitric-oxide Synthase (NOS)-associated Protein Inhibits NOS Dimerization and Activity
Journal of Biological Chemistry, 1999
A variety of transcriptional and post-transcriptional mechanisms regulate the expression of the inducible nitric-oxide synthase (iNOS, or NOS2). Although neurons and endothelial cells express proteins that interact with and inhibit neuronal NOS and endothelial NOS, macrophage proteins that inhibit NOS2 have not been identified. We show that murine macrophages express a 110-kDa protein that interacts with NOS2, which we call NOS-associated protein-110 kDa (NAP110). NAP110 directly interacts with the amino terminus of NOS2, and inhibits NOS catalytic activity by preventing formation of NOS2 homodimers. Expression of NAP110 may be a mechanism by which macrophages expressing NOS2 protect themselves from cytotoxic levels of nitric oxide.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1996
The nitric oxide synthases (NOS-I, neuronal, NOS-II, inducible, and NOS-III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate-liganded protoporphyrin IX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L-arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca(2+)-calmodulin and/...
Distinct Dimer Interaction and Regulation in Nitric-oxide Synthase Types I, II, and III
Journal of Biological Chemistry, 2002
Homodimer formation activates all nitric-oxide synthases (NOSs). It involves the interaction between two oxygenase domains (NOSoxy) that each bind heme and (6R)-tetrahydrobiopterin (H4B) and catalyze NO synthesis from L-Arg. Here we compared three NOSoxy isozymes regarding dimer strength, interface composition, and the ability of L-Arg and H4B to stabilize the dimer, promote its formation, and protect it from proteolysis. Urea dissociation studies indicated that the relative dimer strengths were NOSIIIoxy > > NOSIoxy > NOSIIoxy (endothelial NOSoxy (eNOSoxy) > > neuronal NOSOXY (nNOSoxy) > inducible NOSoxy (iNOSoxy)). Dimer strengths of the full-length NOSs had the same rank order as judged by their urea-induced loss of NO synthesis activity. NOSoxy dimers containing L-Arg plus H4B exhibited the greatest resistance to urea-induced dissociation followed by those containing either molecule and then by those containing neither. Analysis of crystallographic structures of eNOSoxy and iNOSoxy dimers showed more intersubunit contacts and buried surface area in the dimer interface of eNOSoxy than iNOSoxy, thus revealing a potential basis for their different stabilities. L-Arg plus H4B promoted dimerization of urea-generated iNOSoxy and nNOSoxy monomers, which otherwise was minimal in their absence, and also protected both dimers against trypsin proteolysis. In these respects, L-Arg alone was more effective than H4B alone for nNOSoxy, whereas for iNOSoxy the converse was true. The eNOSoxy dimer was insensitive to proteolysis under all conditions. Our results indicate that the three NOS isozymes, despite their general structural similarity, differ markedly in their strengths, interfaces, and in how L-Arg and H4B influence their formation and stability. These distinguishing features may provide a basis for selective control and likely help to regulate each NOS in its particular biologic milieu.
Journal of the American Chemical Society, 1999
Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide. N 5-(1-Iminoethyl)-L-ornithine (L-NIO, 5) is a natural product known to inactivate NOS, but the mechanism of inactivation is unknown. Upon incubation of iNOS with L-NIO a type I binding difference spectrum is observed, indicating that binding at the substrate binding site occurs. L-NIO is shown to be a time-dependent, concentrationdependent, and NADPH-dependent irreversible inhibitor of iNOS with K I and k inact values of 13.7 (1.6 µM and 0.073 (0.003 min-1 , respectively. During inactivation the heme chromophore is partially lost (Figure 1); HPLC shows that the loss corresponds to about 50% of the heme. Inclusion of catalase during incubation does not prevent heme loss. N 5-(1-Imino-2-[ 14 C]ethyl)-L-ornithine (11) inactivates iNOS, but upon dialysis or gel filtration, no radioactivity remains bound to the protein or to a cofactor. The only radioactive product detected after enzyme inactivation is N ω-hydroxy-L-NIO (12); no C ω-hydroxy-L-NIO (13) or N δ-acetyl-L-ornithine (14) is observed (Figure 2). The amount of 12 produced during the inactivation process is 7.7 (0.2 equiv per inactivation event. Incubations of 12 with iNOS show time-, concentration-, and NADPH-dependent inactivation that is not reversible upon dilution into the assay solution. Incubations that include an excess of L-arginine or with substitution of NADP + for NADPH result in no significant loss of enzyme activity. The K I and k inact values for 12 are 830 (160 µM and 0.0073 (0.0007 min-1 , respectively. The magnitude of these kinetic constants (compared with those of 5) suggest that 12 is not an intermediate of L-NIO inactivation of iNOS. Compound 12 also is a substrate for iNOS, exhibiting saturation kinetics with K m and k cat values of 800 (85 µM and 2.22 min-1 , respectively; the product is shown to be N δ-acetyl-L-ornithine (14) (Figure 3). The k cat and k inact values for 12 can be compared directly to give a partition ratio (k cat /k inact) for inactivation of 304; i.e., there are 304 turnovers to give NO per inactivation event. This high partition ratio further supports the notion that 12 is not involved in L-NIO inactivation of iNOS. C ω-Hydroxy-L-NIO (13) is not an inactivator of iNOS. These results suggest that L-NIO inactivation occurs after an oxidation step (NADPH is required for inactivation) but prior to a hydroxylation step (12 and 13 are not involved). Inactivation of iNOS by N 5-(1-imino-2-[ 2 H 3 ]ethyl)-L-ornithine (15) exhibits a kinetic isotope effect on H k inact / D k inact of 1.35 (0.08 and on H (k inact /K I)/ D-(k inact /K I) of 1.51 (0.3, suggesting that the methyl C-H bond is cleaved in a partially rate-determining step prior to hydroxylation, and that leads to inactivation. A new NADPH-dependent 400 nm peak in the HPLC of L-NIO-inactivated iNOS is produced (Figure 4). LC-electrospray mass spectrometry (Figure 5) demonstrates the m/z of the new metabolite to be 583, which is shown to correspond to biliverdin (23) (Figures 6 and 7). Two possible mechanisms for the formation of biliverdin during inactivation are proposed (Schemes 10 and 11). When 14 is incubated with iNOS, time-, concentration-, and NADPH-dependent loss of enzyme activity is observed (K I and k inact values are 490 mM and 0.24 min-1 , respectively); iNOS inactivation by 14 can be prevented by inclusion of L-arginine, but not D-arginine, in the inactivation mixtures, suggesting that the inactivator acts at the arginine binding site. However, 14 is not produced from L-NIO (Figure 2) and, therefore, is not involved in L-NIO inactivation.