Thesis (Ph. D.)--University of Nevada, Reno, 2005. / "August, 2005." Includes bibliographical references. Online version available on the World Wide Web. Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2005]. 1 microfilm reel ; 35 mm.

Carbon Monoxide Dehydrogenase (CODH), also known as Acetyl-CoA synthase (ACS), is one of seven known Ni containing enzymes. CODH/ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl-CoA. Recently, X-ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr) and CODH from Carboxydothermus hydrogenoformans (CODHCh) have been published. These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the β subunit of heterotetrameric α2β2 enzyme from Moorella thermoacetica (CODHMt), formerly Clostridium thermoaceticum. Neither CODHRr nor CODHCh contain an α-subunit as is found in CODHMt. The precise structure of the active site for acetyl-CoA synthase, called the A-cluster, is not known. Therefore, crystallization of the α subunit is required to solve the remaining structural features of CODH/ACS. Obtaining crystals and determining the X-ray crystal structure is a high-risk endeavor, and a second project was pursued involving the preparation, expression and analysis of various site-directed mutants of CODHMt. Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed. Visual inspection of the crystal structure of CODHRr and CODHCh, along with sequence alignments, indicates that there may be separate pathways for proton and electron transfer during catalysis. Mutants of a proposed proton transfer pathway were characterized. Four semi-conserved histidine residues were individually mutated to alanine. Two (His116Mt and His122Mt) were essential to catalysis, while the other two (His113Mt and His119Mt) attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway.

Recent studies suggest that carbon monoxide (CO) is another gas molecule that has similar biological actions as nitric oxide (NO). The purpose of this study is to investigate the relationship between NO and CO in the survival of naïve rat pheochromocytoma PC12 cells. Western blot analysis revealed that all three isoforms of nitric oxide synthase (NOS) exhibited low expression and two isoforms of heme oxygenase (HO), especially HO-1, exhibited higher expression in PC12 cells under basal condition. Exposure of PC12 cells for 24 h to the NO scavenger, carboxy-2-phenyl-4,4,5,5,- tetramethylimidazoline-1-oxy-1-3-oxide (carboxy-PTIO, 2 £gmol) or HO inhibitor, zinc protoporphyrinIX (ZnPP, 25 nmol) resulted in a progressive reduction in mitochondria dehydrogenase activity reflected cell viability as determined by the WST-1 (4-[3-(4-lodophenyl)- 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay. On the other hand, incubation with NO donors, amino-3-morpholinyl- 1,2,3-oxadiazolium chloride (SIN-1, 1 £gmol) or S-Nitroso-N-acetyl- penicillamine (SNAP, 1 £gmol), or the CO precursor, hematin (500 nmol), resulted in an elevation in cell viability. The progressive reduction in cell viability induced by carboxy-PTIO (2 £gmol) or ZnPP (25 nmol) was significantly blunted by co-treatment with SIN-1 (1 £gmol). However, incubation with the NO precursor, L-arginine (L-Arg, 2 £gmol), or the selective inhibitors for nNOS, iNOS or eNOS, N£s-propyl-L-arginine (NPLA, 100 pmol), S-methylisothiourea (SMT, 10 nmol) or N5-1-Iminoethyl-L-ornithine dihydrochloride (L-NIO, 4 nmol) did not significantly alter cell viability. Co-treatment with carboxy-PTIO (2 £gmol) and L-Arg (2 £gmol) was also ineffective. These results suggest that NO or CO contributes to the survival of naïve PC12 cells.

none

Carbon monoxide (CO) has been identified as an endogenous biological messenger in the brain. Heme oxygenase (HO) catalyzes the metabolism of heme to CO and biliverdin. CO has been shown to act as a neurotransmitter and neuronal messenger in the brain. We reported recently that CO was involved in central cardiovascular regulation, modulated the baroreflex, may affect glutamatergic neurotransmission, and metabotropic glutamate receptors (mGluRs) may be coupled to the activation of HO in the nucleus tractus solitarii (NTS) of rats. We also reported previously that adenosine can increase the release of glutamate in the NTS. The present study was designed to investigate the possible interaction of CO, adenosine, and mGluRs groups in the NTS. Male Sprague-Dawley rats were anesthetized with urethane, and blood pressure were monitored intra-arterially. Unilateral microinjection of ascending doses of hemin (0.01 to 3.3 nmol), a heme molecule cleaved by HO to yield CO, produced decreases in blood pressure and heart rate dose-dependently. In addition, similar cardiovascular effects were observed in adenosine (2.3 nmol) and several agonists for mGluRs groups such as DHPG (group ¢¹) (0.03 nmol), APDC (group ¢º) (0.3 nmol)and L-AP4 (group ¢») (0.3 nmol). These cardiovascular effects of hemin were attenuatd by prior administration of the adenosine receptor antagonist DPSPX (0.92 nmol). Similarly, pre-treatment of HO inhibitor ZnPP¢Á or ZnDPBG (1 nmol) also attenuated the depressor and bradycadic effects of adenosine. Among the mGluRs agonists, prior administration of ZnPP¢Á (1 nmol), an inhibitor of HO activity, significantly attenuated the cardiovascular effects of APDC and L-AP4, and failed to prevent the cardiovascular responses of DHPG. These results indicated an interaction between CO and adenosine, and group ¢º and ¢» mGluRs may be coupled to the activation of HO in central cardiovascular regulation.

Gold used to be regarded as catalytically inert until about 20 years ago when it was shown that supported gold clusters < 5 nm in diameter exhibited some unique catalytic properties. Based on this revelation, several studies have demonstrated the feasibility of reactions previously thought of as impossible on gold. The ability of gold to oxidize CO below ambient temperatures at rates higher than conventional CO oxidation catalysts (Pd and Pt) has been shown to hold potentials for technological applications. Extensive past and on-going research are geared towards elucidating the mechanistic details of this reaction. The nature of the active sites, the effect of the supports and the effect of moisture are still debated in literature. I therefore present some experimental results supported with density functional theory calculations to shed additional light on some of the issues concerning gold catalysis in general, and low temperature CO oxidation in particular. Previous studies of the effect of moisture on oxide-supported gold reported that although water promotes CO oxidation on this surface by as much as two orders of magnitude, it is only a spectator molecule on the surface. I present here evidence for strong water-oxygen interactions when water is co-adsorbed with atomic oxygen on Au(111). Impinging a CO beam on the surface co-adsorbed with oxygen and water produces water-enhanced CO oxidation. Based on these results, I propose that CO reacts with hydroxyls formed from water-oxygen interactions to form CO₂, similar to a previous observation on Pt(111). Exposing a Au(111) surface pre-covered with ¹⁶O to isotopically labeled carbon dioxide (C¹⁸O₂) showed that ¹⁶O¹⁸O (m/e = 34) was produced from carbonate formation and decomposition. Estimates of reaction probability and activation energy gave ~ 10⁻⁴ - 10⁻⁵ and -0.15 eV respectively. The effect of annealing on the reactivity of oxygen pre-covered Au(111) was investigated using water, carbon monoxide and carbon dioxide as probe molecules. Precovering Au(111) with atomic oxygen followed by annealing resulted in surfaces that were less reactive towards water, CO and CO₂. Annealing is believed to stabilize the reactive metastable oxygen thereby increasing the barrier to reaction similar to what is reported on other surfaces. / text

We measure inelastic cross sections for atomic thorium (Th) and molecular thorium monoxide (ThO) in collisions with $^3$He at temperatures near 1 K. We determine the Zeeman relaxation cross section for Th ($^3$F$_2$) to be $\sim 2 \times 10^{-17}$~cm$^{-2}$ at 800~mK. We study electronic inelastic processes in Th ($^3$P$_0$) and find no quenching even after $10^6$ collisions at 800~mK. We measure the vibrational quenching cross section for ThO~(X,~$\nu=1$) to be $(7.9 \pm 2.7) \times 10^{-19}$~cm$^{-2}$ at 800~mK. Finally, we observe indirect evidence for ThO (X, $\nu=0$)--$^3$He van der Waals complex formation, and measure the 3-body recombination rate constant to be $\Gamma_3 = (8 \pm 2) \times 10^{-33}$~cm$^6$s$^{-1}$ at 2.4~K. The stability of the ground Th ($^3$F$_2$) state, metastable Th ($^3$P$_0$) state, and vibrational excited ThO (X, $\nu=1$) state provides data on anisotropic interactions in new systems and opens up the possibility for further studies and experiments, including trapping. / Physics

No description available.

No description available.

Carbon Monoxide Dehydrogenase (CODH), also known as Acetyl-CoA synthase (ACS), is one of seven known Ni containing enzymes. CODH/ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl-CoA. Recently, X-ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr) and CODH from Carboxydothermus hydrogenoformans (CODHCh) have been published. These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the β subunit of heterotetrameric α2β2 enzyme from Moorella thermoacetica (CODHMt), formerly Clostridium thermoaceticum. Neither CODHRr nor CODHCh contain an α-subunit as is found in CODHMt. The precise structure of the active site for acetyl-CoA synthase, called the A-cluster, is not known. Therefore, crystallization of the α subunit is required to solve the remaining structural features of CODH/ACS. Obtaining crystals and determining the X-ray crystal structure is a high-risk endeavor, and a second project was pursued involving the preparation, expression and analysis of various site-directed mutants of CODHMt. Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed. Visual inspection of the crystal structure of CODHRr and CODHCh, along with sequence alignments, indicates that there may be separate pathways for proton and electron transfer during catalysis. Mutants of a proposed proton transfer pathway were characterized. Four semi-conserved histidine residues were individually mutated to alanine. Two (His116Mt and His122Mt) were essential to catalysis, while the other two (His113Mt and His119Mt) attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway.