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Nitric oxide synthase (NOS) catalyses the production of nitric oxide (NO). A cytochrome P450-like oxygenase, it uses two monooxygenation steps to convert L-arginine (L-arg) first to N -hydroxy-L-arginine (NOHA), a stable intermediate, and then to L-citrulline and NO. Mammalian NOSs are homodimeric enzymes. Each monomer is composed of an oxygenase domain (containing the L-arg binding site, a heme ligated by a cysteine thiolate, and a tetrahydrobiopterin (H4B)) and a reductase domain (binding NADPH, FAD, and FMN). NOS substrates are O2, L-arg, and NADPH. NADPH is the source of electrons required for oxygen activation. H4B is a vital cofactor that aids dimerisation and acts as a reducing/oxidising agent. Controversy still exists as to the final oxygenating species in the NOS mechanism, but the general reaction scheme is known. The ferric heme is reduced to the ferrous state by an electron from the reductase domain. Then oxygen binds to form the oxy-ferrous species. Then H4B donates an electron to form a peroxy-ferric species. It is likely this then forms a compound 1 (Fe(IV)+.=O) species that is the final oxygenating species. This thesis probes the mechanism of NOS to further define the mechanistic intermediates involved. The role of H4B in NO synthesis has been probed in both normal turnover conditions and special case reactions. To elucidate this mechanism further a mutant with a residue capable of stabilising the activated oxygen species was created, G586S, where glycine 586 of nNOS was replaced with a serine. This serine was within hydrogen bonding distance of the oxy-heme. A stabilised intermediate was observed by stopped flow reaction in the presence of H4B, but not aH4B (an inactive pterin analogue). Here single turnover reactions, each following either the reaction of L-arg to NOHA or NOHA to citrulline, were performed on the mutant using an external source of electrons. The reaction products were observed by HPLC. The mutant appears capable of the conversion of NOHA to citrulline, but not L-arg to NOHA. The WT enzyme appears capable of both. The intermediate is observed with either L-arg or NOHA bound, suggesting both reactions proceed via the same active oxygenating species. The inability of the mutant to catalyse the conversion of L-arg to NOHA may be due to protonation of the substrate hindering reaction such that the active oxygenating species decays before reaction can occur. This mutation, in allowing separation of the two monooxygenation steps, deserves further study. H4B binds at the dimer interface of NOS. Here the -systems of the pterins are only 13Å apart. This is within allowed distances for efficient electron transfer. Electron transfer between hemes, via the pterins, would allow a route for the breakdown of a dead end, ferrous-NO, species. Stopped flow monitoring of the decay of the ferrous-heme NO complex with nNOSoxy dimers with varying proportions of the hemes in the ferrous heme-NO complex showed no electron transfer between hemes of the dimer. The rate of decay of the ferrous heme-NO complex in oxygenated buffer is 0.12 s-1 for all conditions tested here. H4B-deficiency leads to several diseases. H4B makes a poor drug due to instability and cost, the search for druggable analogues of it is ongoing. H4B analogues blocked at the 6,7-positions in the dihydropterine-form have been screened here for catalytic activity. Several have shown comparable ability to catalyse NO production in vitro. Structure function analysis of these analogues has revealed the extent extension is tolerated at the C6 and C7 positions of the pterin.

Current and future pollutant enussion legislation calls for decreased NOx emissions from combustion systems. A review of techniques used for NOx abatement led to the choice of combustor redesign to be the most cost effective method available. This led to the design, construction and development of a combustion system that utilised a Coanda ejector to generate recirculation of the exiting high temperature combustion products to mix with the air supply. Cooling of the burner was integrated into the design through the use of the air and fuel supplies. Computational fluid dynamics was used to model and aid development of the design. The model was used to predict NOx and CO emissions and the fuel-air mixing pattern. This, along with an analysis of experimental results and observations led to an understanding of the burner operation with respect to pollutant emissions and stability. NOx emissions from the Coanda burner were found to be lowest when using a 0.2 mm Coanda gap width, resulting in 16 ppm NOx being emitted at an air to fuel ratio of 1.5. However, the use ofa 0.2 mm Coanda gap width required an air supply pressure of up to 4 bar. The use of a 0.5 mm Coanda gap width enabled burner operation at lower air supply pressures. The resulting NOx emissions were measured as 23 ppm at an air to fuel ratio of 1.I, with a corresponding exit gas temperature of 2200 K. Flue gas recirculation quantity, flame stability, flame stabiliser shape and operational limits proved to be inter-linked in the reduction of NOx emissions. It was found that fuel-air mixing was controlled by the entrainment properties of the Coanda ejector and the flame stabiliser. The average oxygen concentration entering the combustion chamber when using a 0.2 mm and 0.5 mm Coanda gap width was 13.7 % and 16.6 %, respectively. Due to the position of the fuel injector, a fuel rich region formed behind the flame stabiliser. With a suitable flame stabiliser geometry and the use of 'fingers', low NOx combustion and flame stability was achieved near stoichiometric conditions. It was shown that the design of the burner enabled very low pollutant emissions near stoichiometric conditions, resulting in high exit gas temperatures. Conceivable applications of this type of burner could lie in small and intermediate furnaces where low NOx emissions are required. Additionally, very high temperature applications, such as glass furnaces could benefit in both cost and pollutant emissions from such a burner.

Spectrophotometric (including stopped-flow) techniques were used to examine the kinetics of NO-group transfer reactions (transnitrosation) between S-nitrosothiols (RSNO) and a wide range of sulfur/nitrogen nucleophiles in aqueous solution. A metal-ion chelator was added in all experiments to prevent RSNO decay and NO liberation catalysed by copper ions. In most cases reaction was envisaged as rate- determining attack of the nucleophile at the nitrogen atom of the -SNO moiety, and hence S-nitrosothiols essentially acted as electrophilic nitrosating agents. Sulfite, thiosulfate, thiourea, thiocyanate and thiomethoxide, were sufficiently nucleophilic to induce nitrosothiol decomposition at physiological pH. Reaction with sulfide (pH > 7.4) afforded the orange-yellow anion, SSNO, and embodies a potential quantitative test for RSNOs. S-Nitrosopenicillamine was reactive enough to allow a thorough investigation into its reaction at basic pH with primary, secondary (creating carcinogenic N-nitrosamines), and tertiary amines, as well as ambident (e.g. thiomorpholine) and alpha nucleophiles (e.g. azide ion). Parallels could be made with analogous studies using other nitroso compounds such as MNTS. The generality of the reaction of a S-nitrosothiol with a large excess of the corresponding or a different thiol was also assessed. Ammonia and not nitric oxide was confirmed as the primary nitrogenous product of this highly complicated process. Mechanistic details of the copper(I) catalysed decomposition of some novel S- nitroso derivatives (e.g. a synthesised S-nitroso-1 -thiosugar) are reported. The two- stage degradation pathway involved an initial Cu(^+) promoted component that halted at incomplete conversion, and was accompanied by a large thermal reaction. An explanation of this unique pattern has been offered in terms of the generation of a disulfide-Cu(^2+) complex, in which copper is/is not accessible to reduction.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Graduation date: 1995

Introduction: To evaluate the effectiveness of smoking cessation intervention, reliable outcome is essential. Exhaled nitric oxide (ENO) is decreased in smokers, tends to normalize after cessation and might be a good tool to evaluate abstinence. Objective: To evaluate changes in ENO after smoking abstinence of 7 or more days. Methods: 58 smokers in a cessation attempt and 12 non-smokers were recruited: 7 visits for smokers and 2 for non-smokers. Carbon monoxide and cotinine were used to detect smoking status. Results: ENO is decreased in smokers compared to non-smokers (10.8 vs. 20.1 ppb, p<0.001). There was no significant difference in ENO pre and post quitting (p=0.080) although there was a trend to increase as early as 3 days after abstinence (10.78 vs. 15.11, p>0.05). There were no differences in nasal NO measurements (p=0.278). Conclusion: ENO doesn’t seem to be a reliable marker of short-term abstinence.

Introduction: To evaluate the effectiveness of smoking cessation intervention, reliable outcome is essential. Exhaled nitric oxide (ENO) is decreased in smokers, tends to normalize after cessation and might be a good tool to evaluate abstinence. Objective: To evaluate changes in ENO after smoking abstinence of 7 or more days. Methods: 58 smokers in a cessation attempt and 12 non-smokers were recruited: 7 visits for smokers and 2 for non-smokers. Carbon monoxide and cotinine were used to detect smoking status. Results: ENO is decreased in smokers compared to non-smokers (10.8 vs. 20.1 ppb, p<0.001). There was no significant difference in ENO pre and post quitting (p=0.080) although there was a trend to increase as early as 3 days after abstinence (10.78 vs. 15.11, p>0.05). There were no differences in nasal NO measurements (p=0.278). Conclusion: ENO doesn’t seem to be a reliable marker of short-term abstinence.

Calmodulin (CaM) is a calcium-binding protein that has promiscuous regulatory interactions with over three hundred intracellular protein targets. The focus of this study was to characterize the functional role of phosphorylated CaM in vitro and calcium-deficient CaM (Apo-CaM) ex vivo. In the in vitro study, the effect of phosphorylated CaM on the binding and activation of CaM target proteins was analyzed using mammalian Nitric Oxide Synthase (NOS). NOS is an enzyme that catalyzes the conversion of L-arginine to L-citrulline and •NO. In addition, the activation of NOS by modified CaM proteins was also analyzed in the presence of a CaM binding peptide, PEP-19. Protein trafficking experiments were performed ex vivo to extend our understanding of Apo-CaM’s functional role in mammalian cells. The cell lines that were used in this investigation include mouse Embryonic Stem Cells (mESC), Human Umbilical Vein Endothelia Cells (HUVEC) and Human Neuronal Glioma Cells (HNGC). The major finding of this projects are: phosphorylation of selective CaM residues can attenuated NOS activity, electrostatic interactions are important in the activation of iNOS by CaM, and the activation of iNOS by CaM occurs in a calcium-dependent manner

Calmodulin (CaM) is a calcium-binding protein that has promiscuous regulatory interactions with over three hundred intracellular protein targets. The focus of this study was to characterize the functional role of phosphorylated CaM in vitro and calcium-deficient CaM (Apo-CaM) ex vivo. In the in vitro study, the effect of phosphorylated CaM on the binding and activation of CaM target proteins was analyzed using mammalian Nitric Oxide Synthase (NOS). NOS is an enzyme that catalyzes the conversion of L-arginine to L-citrulline and •NO. In addition, the activation of NOS by modified CaM proteins was also analyzed in the presence of a CaM binding peptide, PEP-19. Protein trafficking experiments were performed ex vivo to extend our understanding of Apo-CaM’s functional role in mammalian cells. The cell lines that were used in this investigation include mouse Embryonic Stem Cells (mESC), Human Umbilical Vein Endothelia Cells (HUVEC) and Human Neuronal Glioma Cells (HNGC). The major finding of this projects are: phosphorylation of selective CaM residues can attenuated NOS activity, electrostatic interactions are important in the activation of iNOS by CaM, and the activation of iNOS by CaM occurs in a calcium-dependent manner