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No description available.

No description available.

<p> Nitrate assimilation is the process by which nitrate is converted into ammonia, and ultimately into organic nitrogenous compounds, which are then made available to organisms which require an exogenous supply of organic nitrogen. Nitrite is an intermediate in this process and the mechanism of its conversion to ammonia, which is catalyzed by the enzyme nitrite reductase, needs clarification. </p> <p> The purpose of this investigation was to find a suitable assay system for nitrite reductase in N. crassa and to examine some aspects of the metabolic control of the enzyme. A new assay system for nitrite reductase is described and evidence suggesting that the enzyme is derepressible is presented. </p> / Thesis / Master of Science (MSc)

<p> Nitrite is an intermediate in the pathway of nitrate assimilation. Several questions about reduction beyond the level of nitrite remain to be answered. </p> <p> The purpose of this investigation was to induce mutants deficient in nitrite reduction and to characterize these mutants phenotypically and genetically in an attempt to answer the following questions: </p> <p> (a) How many enzymes are required in vivo for nitrite reduction? </p> <p> (b) How many genes control nitrite reduction? </p> <p> (c) Is nitrite reductase localized within a particle? </p> <p> The results of this investigation showed that nitrite reductase is controlled by at least three genes and three cistrons on two linkage groups. None of the 'nitrite-mutants' were allelic with nitrate reductase mutants. There appeared to be more than one type of nitrite reductase activity in extracts of repressed wild type mycelia. Only one of these nitrite reductase species seemed to be necessary for the reduction of nitrite in vivo. </p> / Thesis / Master of Science (MSc)

Multi-electron multi-proton reactions form the basis of nearly every chemical reaction involved in energy storage and manipulation. Despite their importance, the basic properties of these chemical transformations, such as the details of how electron transfer and proton-coupled redox events that must occur during these reactions are controlled, remain poorly understood. The sulfite and nitrite reductase family of enzymes are responsible for carrying out the six-electron reduction of sulfite to sulfide and nitrite to ammonia, respectively. These enzymes play fundamental roles in microbial metabolism and are either dissimilatory or assimilatory in nature. Multi-electron multi-proton reactions are investigated by the study of the catalytic mechanisms of two enzymes that are structurally different, but carry out similarly complex chemistry: the dimeric multi heme cytochrome c nitrite reductase from Shewanella oneidensis and the monomeric siroheme and [4Fe-4S] cluster containing sulfite reductase from Mycobacterium tuberculosis. Employing protein electrochemistry the properties of electron transfer steps and proton-coupled redox steps that occur throughout the catalytic cycle of cytochrome c nitrite reductase during its reduction of substrate revealed the strategies employed by this enzyme. The results presented indicate the reduction of substrate by the enzyme occurs in a series of one electron steps rather than coupled two-electron transfers. Mutational analysis of active site amino acids reveals their role in governing proton coupled redox events, which likely involves a hydrogen bonding network consisting of these residues and water molecules. Additionally, steady state kinetics assays coupled to site-directed mutagenesis of M. tuberculosis sulfite reductase identify a tyrosine residue adjacent to the active site which partially controls substrate preference, by influencing the electronic environment of the active site siroheme cofactor. Stopped-flow absorbance spectroscopy and rapid freeze quench electron paramagnetic resonance studies provide a first glimpse of a potential reaction intermediate during reduction of sulfite by sulfite reductase. Overall, our fundamental understanding of how sulfite and nitrite reductase enzymes catalyze complex multi-electron multi-proton reactions is advanced, and insight into the different approaches Nature employs to govern such powerful chemistry is revealed.

Neisseria meningitidis is the causative agent of potentially life-threatening meningitis and septicaemia. According to W.H.O., meningococcal disease causes at least 500,000 cases and results in 50,000 deaths worldwide each year (W.H.O., 2008). Neisseria gonorrhoeae is causing the second most common sexually transmitted bacterial infection, with a global incidence of 62 million cases per year. Previous studies have shown surface expressed proteins like pilin, the subunit protein that forms pili (Type IV Fimbriae), in N. meningitidis and N. gonorrhoeae are post-translationally modified by O-glycosylation. This modification has been proposed to be of importance in the pathogenesis of these species. Although the exact function of these post-translational modifications are not fully understood, it is suggested that these modification have a role for immune evasion in the host. In this thesis, an additional outer membrane glycoprotein was identified in pathogenic Neisseria, the nitrite reductase AniA. Mass spectrometry analysis showed that AniA is glycosylated in its C-terminal imperfect (AASAP) repeat region by the pilin glycosylation pathway. This is the first report of a general O-glycosylation pathway in a prokaryote. It was shown AniA is surface exposed. To investigate whether AniA is subject to immune selection, a large collection of N. meningitidis and N. meningitidis clinical isolates were sequence analysed and evaluated. Analysis of published AniA 3D structure revealed that AniA displayed polymorphisms in residues that map to the surface of the protein. This suggests that AniA is under immune selection, and that glycosylation may facilitate immune evasion. Sequencing analyses revealed a frame shift mutation that abolished AniA expression in 34% of N. meningitidis strains surveyed. However, all N. gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology. In summary, the data presented here suggested that the protein may be under immune selective pressure. The addition of a phase variable glycan to this surface protein may serve as an additional immune evasion strategy. Immune selection on surface proteins in these host-adapted pathogens may have been the driving force for the evolution of this general O-glycosylation pathway. Therefore, the discovery that AniA is a glycoprotein has given insights into the pathogenesis and the host-pathogen interactions of these organisms.

Membrane inlet mass spectrometry was used to observe nitric oxide in the well-studied reaction of nitrite with hemoglobin. The membrane inlet was submerged in the reaction solutions and measured NO in solution via its flux across a semipermeable membrane leading to the mass spectrometer detecting the mass-to-charge ratio m/z 30. This method measures NO directly in solution and is an alternate approach compared with methods that purge solutions to measure NO. Addition to deoxy-Hb(FeII) (near 38 μM heme concentration) of nitrite in a range of 80 μM to16 mM showed no accumulation of either NO or N2O3 on a physiologically relevant time scale with a sensitivity near 1 nM. The addition of nitrite to oxy-Hb(FeII) and met-Hb(FeIII) did not accumulate free NO to appreciable extents. These observations show that for several minutes after mixing nitrite with hemoglogin, free NO does not accumulate to levels exceeding the equilibrium level of NO. The presence of cyanide ions did not alter the appearance of the data; however, the presence of 2 mM mercuric ions at the beginning of the experiment with deoxy-Hb(FeII) shortened the initial phase of NO accumulation and increased the maximal level of free, unbound NO by about twofold. These experiments appear consistent with no role of met-Hb(FeIII) in the generation of NO and an increase in nitrite reductase activity caused by the presumed binding of mercuric to cysteine residues. These results raise questions about the ability of reduction of nitrite mediated by deoxy-Hb(FeII) to play a role in vasodilation.

It is usually assumed that the reduction of nitrite by nitrite reductase results in the formation of ammonia. The purpose of this investigation was to enquire whether more than one nitrite reductase activity is responsible for in vivo nitrite reduction. An assay system which measures the production of ammonia, as a result of nitrite reductase is described. The. reduction of nitrite by nitrite reductase did not result in the formation of stoichiometric amounts of ammonia. Nitrite non-utilizing mutants showed that nitrite reduction could occur in vitro with no subsequent formation of ammonia or could result in the formation of essentially stoichiometric amounts of ammonia. Sedimentation velocity gradient centrifugation resulted in the separation of at least two peaks of nitrite reductase activity. A model is described which accounts for the results in terms of two nitrite reductase activities, necessary for in vivo nitrite reduction. / Thesis / Master of Science (MSc)

The need for an alternative to red cells in transfusions has led to the creation of hemoglobin-based oxygen carriers (HBOCs). However, evaluations of all products tested in clinical trials have noted cardiovascular complications, raising questions about their safety that led to the abandonment of all those products. It has been considered that the adverse side effects come from the scavenging of the vasodilator – nitric oxide (NO) by the deoxyheme sites of the hemoglobin derivatives. Another observation is that HBOCs with lower oxygen affinity than red cells release oxygen prematurely in arterioles, triggering an unwanted homeostatic response. Since the need for such a product remains critical, it is important to understand the reactivity patterns that contribute to the observed complications. Various alterations of the protein have been attempted in order to reduce HBOC-induced vasoconstriction. Recent reports suggest that a safe and effective product should be pure, homogenous and have a high molecular weight along with appropriate oxygenation properties. While these properties are clearly important, vasodilatory features of hemoglobin through its nitrite reductase activity may also act as an in situ source of NO. It follows that HBOCs with an enhanced ability to produce NO from endogenous nitrite may serve to counteract vasoactivity associated with NO-scavenging by hemoglobin. Here we characterize the effects of different protein modifications on the nitrite reductase activity of hemoglobin. We produced a variety of HBOCs that include cross-linked tetramers, polyethylene glycol (PEG) conjugates and bis-tetramers of hemoglobin. We report that the rate of NO production strongly depends on the conformational state of the protein, with R-state stabilized proteins (PEG-Hbs), exhibiting the fastest rates. In particular, we found that PEGylated bis-tetramers of hemoglobin (BT-PEG) exhibit increased nitrite reductase activity while retaining cooperativity and stability. Animal studies of BT-PEG demonstrated that this material is benign: it did not cause significant increases in systemic blood pressure in mice, the major side effect associated with existing HBOCs. BT-PEG exhibits an enhanced nitrite reductase activity together with sample purity and homogeneity, molecular size and shape, and appropriate oxygenation properties, characteristics of a clinically useful HBOC.