Our long-term objectives are to understand the functions of vitamin B 12 compounds in enzymatic reactions and as therapeutic agents in terms of their chemical properties. Some specific goals of this research project are to investigate the interactions of nitrogen oxides, NO and N20, and nitrite anion with vitamin B12 (cobalamin) compounds in vitro in order to understand their effects on enzymatic reactions of methionine synthase, B 12-MS, and their effectiveness for sequestering trauma induced NO in vivo. One hypothesis is that redox reactions of NO and N20 with the Co(II) and Co(I) forms of cobalamin can effect enzyme functioning. Specifically, we plan to study the complexes formed and the redox behavior of systems of NO, N20, and the nitrite anion with various cobalamin compounds in their three cobalt oxidation states using electrochemical measurements, UV-VIS spectroscopy, resonance Raman spectroscopy (RRS), surface enhanced resonance Raman spectroscopy (SERRS), and electron paramagnetic resonance (EPR) spectroscopy. Nitrogen species formed as reaction products will be investigated by gas chromatography-mass spectrometry (GC-MS), We will also study the reductive cleavage mechanism for the activation of methylcobalamin and adenosylcobalamin. Our aim is to measure equilibrium binding constants, redox reaction rates, and electrochemical reduction potentials for cobalamin redox couples and also to determine the structure of NO binding in their nitrosyl complexes. Experiments will also investigate the solvent and pH effects on the equilibrium and kinetic properties of the complexes. In addition, we will investigate the inactivation of pure methionine synthase obtained from a recombinant strain of E. Coil which overproduces the enzyme. We will compare NO and N20 inactivation by various means including deactivation in an electrochemical cell which produces the cob(I)alamin form of the enzyme, analysis of the inactivated enzyme by twodimensional polyacrylamide gel electrophoresis, and stopped-flow kinetic measurements of enzyme catalysis in the presence of NO and N20. Additionally, we will correlate our chemical studies with the effect of various cobalamin compounds on NO generated at the surface of endothelium cells in physiological experiments to be done at Ohio University. These results should help in understanding how NO and N20 work in inhibiting B 12-MS function, if NO can act as a regulator for MS-B 12, and how cobalamins can detoxify organisms with elevated levels of NO which cause the condition of hypotension.