Luminescent Ruthenium(II)− and Rhenium(I)−Diimine Wires Bind Nitric Oxide Synthase

Ru(II)− and Re(I)−diimine wires bind to the oxygenase domain of inducible nitric oxide synthase (iNOSoxy). In the ruthenium wires, [Ru(L)_2L‘]^(2+), L‘ is a perfluorinated biphenyl bridge connecting 4,4‘-dimethylbipyridine to a bulky hydrophobic group (adamantane, 1), a heme ligand (imidazole, 2), or F (3). 2 binds in the active site of the murine iNOSoxy truncation mutants Δ65 and Δ114, as demonstrated by a shift in the heme Soret from 422 to 426 nm. 1 and 3 also bind Δ65 and Δ114, as evidenced by biphasic luminescence decay kinetics. However, the heme absorption spectrum is not altered in the presence of 1 or 3, and Ru−wire binding is not affected by the presence of tetrahydrobiopterin or arginine. These data suggest that 1 and 3 may instead bind to the distal side of the enzyme at the hydrophobic surface patch thought to interact with the NOS reductase module. Complexes with properties similar to those of the Ru−diimine wires may provide an effective means of NOS inhibition by preventing electron transfer from the reductase module to the oxygenase domain. Rhenium−diimine wires, [Re(CO)_3L_1L_1‘]+, where L_1 is 4,7-dimethylphenanthroline and L_1‘ is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole to a distal imidazole (F_8bp-im) (4) or F (F_9bp) (5), also form complexes with Δ114. Binding of 4 shifts the Δ114 heme Soret to 426 nm, demonstrating that the terminal imidazole ligates the heme iron. Steady-state luminescence measurements establish that the 4:Δ114 dissociation constant is 100 ± 80 nM. Re−wire 5 binds Δ114 with a K_d of 5 ± 2 μM, causing partial displacement of water from the heme iron. Our finding that both 4 and 5 bind in the NOS active site suggests novel designs for NOS inhibitors. Importantly, we have demonstrated the power of time-resolved FET measurements in the characterization of small molecule:protein interactions that otherwise would be difficult to observe

Oxygenase Domain of Drosophila melanogaster Nitric Oxide Synthase: Unique Kinetic Parameters Enable a More Efficient NO Release

Although nitric oxide (NO) is important for cell signaling and nonspecific immunity in the fruit fly Drosophila melanogaster, little is known about its single NO synthase (dNOS). We expressed the oxygenase domain of dNOS (dNOSoxy), characterized its spectroscopic, kinetic, and catalytic properties, and interpreted them in light of a global kinetic model for NO synthesis. Single turnover reactions with ferrous dNOSoxy showed it could convert Arg to N'omega-hydroxy-l-arginine (NOHA), or NOHA to citrulline and NO, when it was given 6R-tetrahydrobiopterin and O2. The dNOSoxy catalyzed Arg hydroxylation and NOHA oxidation at rates that matched or exceeded the rates catalyzed by the three mammalian NOSoxy enzymes. Consecutive heme-dioxy, ferric heme-NO, and ferric heme species were observed in the NOHA reaction of dNOSoxy, indicating that its catalytic mechanism is the same as in the mammalian NOS. However, NO dissociation from dNOSoxy was 4 to 9 times faster than that from the mammalian NOS enzymes. In contrast, the dNOSoxy ferrous heme-NO complex was relatively unreactive toward O2 and in this way was equivalent to the mammalian neuronal NOS. Our data show that dNOSoxy has unique settings for the kinetic parameters that determine its NO synthesis. Computer simulations reveal that these unique settings should enable dNOS to be a more efficient and active NO synthase than the mammalian NOS enzymes, which may allow it to function more broadly in cell signaling and immune functions in the fruit fly

Lipopolysaccharides of brucella abortus and brucella melitensis induce nitric oxide synthesis in rat peritoneal macrophages

Producción CientíficaSmooth lipopolysaccharide (S-LPS) and lipid A of Brucella abortus and Brucella melitensis induced the production of nitric oxide (NO) by rat adherent peritoneal cells, but they induced lower levels of production of NO than Escherichia coli LPS. The participation of the inducible isoform of NO synthase (iNOS) was confirmed by the finding of an increased expression of both iNOS mRNA and iNOS protein. These observations might help to explain (i) the acute outcome of Brucella infection in rodents, (ii) the low frequency of septic shock in human brucellosis, and (iii) the prolonged intracellular survival of Brucella in humans.This work was supported by (grants FIS 96/1017, SAF96-0144, and SAF98-0176

Decreased systemic bioavailability of L-arginine in patients with cystic fibrosis

BACKGROUND: L-arginine is the common substrate for nitric oxide synthases and arginases. Increased arginase levels in the blood of patients with cystic fibrosis may result in L-arginine deficiency and thereby contribute to low airway nitric oxide formation and impaired pulmonary function. METHODS: Plasma amino acid and arginase levels were studied in ten patients with cystic fibrosis before and after 14 days of antibiotic treatment for pulmonary exacerbation. Patients were compared to ten healthy non-smoking controls. RESULTS: Systemic arginase levels measured by ELISA were significantly increased in cystic fibrosis with exacerbation compared to controls (17.3 ± 12.0 vs. 4.3 ± 3.4 ng/ml, p < 0.02). Arginase levels normalized with antibiotic treatment. Plasma L-arginine was significantly reduced before (p < 0.05) but not after treatment. In contrast, L-ornithine, proline, and glutamic acid, all downstream products of arginase activity, were normal before, but significantly increased after antibiotic therapy. Bioavailability of L-arginine was significantly reduced in cystic fibrosis before and after exacerbation (p < 0.05, respectively). CONCLUSION: These observations provide further evidence for a disturbed balance between the L-arginine metabolic pathways in cystic fibrosis

Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

<p>Abstract</p> <p>Background</p> <p>Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1^stand 2^ndEF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.</p> <p>Results</p> <p>The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca²⁺binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10⁵and 5.0 × 10²M^-1with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1^stnon-canonical EF-hand is largely enhanced by the binding of Ca²⁺to the 2^ndcanonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca²⁺binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg²⁺-binding in NCaBD.</p> <p>Conclusions</p> <p>We demonstrated that the 1^stnon-canonical EF-hand of NOX5 has very weak Ca²⁺binding affinity compared with the 2^ndcanonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca²⁺binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca²⁺specific.</p> <p>Graphical abstract</p> <p><display-formula><graphic file="1752-153X-6-29-i1.gif"/></display-formula></p

Dimethylarginine dimethylaminohydrolase I enhances tumour growth and angiogenesis

Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase I. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis in vitro. Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development. Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production