Purification of the terminal oxidases of the facultative anerobe, M. denitrificans, was undertaken. The bacterium contains both a cytochrome c oxidase and a cyanide-insensitive DPNH oxidase. A procedure for purification of each of these enzymes is given. These procedures involve fractionation with ammonium sulfate, acetone, and calcium phosphate gel. By the use of the procedures given, cytochrome c oxidase can be purified seventeen-fold and the DPNH oxidase ninty-fold over the original cell-free extract. The cytochrome c oxidase was found to be associated with the particulate material of the cell, had an optimum activity at pH 7.0 to 7.4, and was not affected by aluminum, magnesium, or manganous ion. The enzyme was completely inhibited by cyanide and carbon monoxide but only 65% by azide. The cytochrome c oxidase oxidized reduced mammalian cytochrome c directly. Oxidative phosphorylation was demonstrated during oxidation of reduced mammalian cytochrome c by cell-free extracts of the bacteria. The absorption maxima of the reduced spectrum of the partially purified cytochrome c oxidase were at 420, 522, and 554 mμ., and the maxima of the difference spectrum were at 427, 522, and 551 mμ. The DPNH oxidase appears to be a soluble flavoprotein. An active DPNH oxidase preparation which was inactivated by ammonium sulfate fractionation, could be reactivated by addition of flavin mononucleotide; flavin adenine dinucleotide restored only partial activity. The Michaelis constant with respect to DPNH of the partially purified DPNH oxidase was found to be 3.42 x 10^-6 moles/liter. The activation energy of the DPNH oxidase was determined and found to be 14,900 cals/mole. The oxygen uptake of a cell-free extract of M. denitrificans functioning as a cytochrome c oxidase and as a DPNH oxidase, was 7.54 u liters/minute and 3.52 u liters/minute respectively. The cytochrome c oxidase appears to be the primary terminal oxidase, however, the DPNH oxidase does make a significant contribution to the aerobic respiration of the bacterium. The participation of the bacterial cytochrome c oxidase and DPNH oxidase in the aerobic respiration of the bacterium is discussed.

No description available.

No description available.

Many oxidoreductases need nicotinamide cofactors for their reactions. The big obstacle of using these syntheses in industry is the high cost of these nicotinamide cofactors. The work here is about finding novel NADH oxidases from Lactococcus lactis and applying in a cofactor regeneration system with carbonyl reductase or alcohol dehydrogenase. NADH oxidases are useful biocatalysts for regenerating nicotinamide cofactors of biological redox reactions. The annotated alkyl hydroperoxide reductase (AhpR) and the H2O-forming enzyme (nox-2) genes from Lactococcus lactis (L. lactis, L.lac-Nox2) were cloned and proteins were expressed and characterized. They were compared with the H2O-former from Lactobacillus sanfranciscensis (L. sanfranciscensis, L.san-Nox2). AhpR is composed of H2O2-forming NADH oxidase (nox-1) and peroxidase and the net reaction of AhpR is the same as nox-2 when oxygen is the substrate. Both nox-1 and nox-2 are flavoproteins and turnover-limited. In the absence of exogenously added thiols, both nox-1 and nox-1/peroxidase are considerably more stable against overoxidation than nox-2. L.san-Nox2 was crystallized and was found to have ADP ligand, but according to the HPLC results, no ADP ligand was found in the L. lac-Nox-2. Enzyme membrane reactor was used for the application of oxidative reaction of cyclohexanol to cyclohexanone, with isolated enzymes horse liver alcohol dehydrogenase and L.lac-Nox2.

Despite a number of kinetic and spectroscopic studies, the chemical mechanisms of amine oxidation by flavoenzymes remain widely debated. The mechanisms of by Nmethyltryptophan oxidase (MTOX) and tryptophan 2-monooxygenase (TMO) were probed using a combination of pH and primary deuterium, solvent, and 15N kinetic isotope effects. Slow substrates were chosen for these studies; MTOX was characterized with N-methylglycine and TMO was characterized with L-alanine. Primary deuterium kinetic isotope effects of 7.2 and 5.3 were observed for sarcosine oxidation by MTOX and for alanine oxidation by TMO, respectively, independent of the substrate concentration and pH. Monitoring the reduction of flavin spectroscopically revealed no intermediate flavin species with both enzyme-substrate systems. Furthermore, the magnitudes of the 15N kinetic isotope effects observed with both systems suggest that nitrogen rehybridization and C-H bond cleavage are concerted. These results are consistent with both enzymes utilizing a hydride transfer mechanism for amine oxidation. The role of the iron ligands of tyrosine hydroxylase (TyrH) was also investigated. TyrH contains one iron per monomer, which is held by three conserved amino acid residues, two histidines and a glutamate. As a probe of the plasticity of the metal binding site, each of the metal ligands in TyrH was substituted with glutamine, glutamate, or histidine. The resulting proteins were characterized for metal content, catalytic activity, and dopamine binding. The H336E and H336Q enzymes retain substantial catalytic activity. In contrast, the E376Q enzyme retains about 0.4% of the wild-type catalytic activity, and the E376H enzyme has no significant activity. The H331E enzyme oxidizes tetrahydropterin in a tyrosine-independent manner. The position of the charge-transfer absorbance band for the H336E and H336Q enzyme-inhibitor complexes is shifted relative to that of the wild-type enzyme, consistent with the change in the metal ligand. In contrast, the E376H and E376Q enzymes catalyze dopamine oxidation. These results provide a reference point for further structural studies of TyrH and the other aromatic amino acid hydroxylases, and for similar studies of other enzymes containing this ironbinding motif.

No description available.

Thesis (Ph. D.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Michigan State University. Dept. of Neuroscience, 2008. / Title from PDF t.p. (viewed on July 23, 2009) Includes bibliographical references. Also issued in print.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Bruce Banks; submitted to the Dept. of Chemistry. Title from PDF t.p. (viewed Jul. 31, 2009). Includes bibliographical references (p. 95-102).

Thesis (Ph.D.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Jo, Hanjoong; Committee Member: Griendling, Kathy; Committee Member: Harrison, David; Committee Member: Wang, May; Committee Member: Yoganathan, Ajit.