title: Mechanistic studies on the reaction between cob(II)alamin and peroxynitrite: evidence for a dual role for cob(II)alamin as a scavenger of peroxynitrous acid and nitrogen dioxide.
abstract: Peroxynitrite/peroxynitrous acid (ONOO(-)/ONOOH; pK(a(ONOOH)) =6.8) is implicated in multiple chronic inflammatory and neurodegenerative diseases. Both mammalian B(12)-dependent enzymes are inactivated under oxidative stress conditions. We report studies on the kinetics of the reaction between peroxynitrite/peroxynitrous acid and a major intracellular vitamin B(12) form, cob(II)alamin (Cbl(II)), using stopped-flow spectroscopy. The pH dependence of the reaction is consistent with peroxynitrous acid reacting directly with Cbl(II) to give cob(III)alamin (Cbl(III)) and (.)NO(2) , followed by a subsequent rapid reaction between (.)NO(2) and a second molecule of Cbl(II) to primarily form nitrocobalamin. In support of this mechanism, a Cbl(II)/ONOO(H) stoichiometry of 2:1 is observed at pH 7.35 and 12.0. The final major Cbl(III) product observed (nitrocobalamin or hydroxycobalamin) depends on the solution pH. Analysis of the reaction products in the presence of tyrosine-a well-established (.)NO(2) scavenger-reveals that Cbl(II) reacts with (.)NO(2) at least an order of magnitude faster than tyrosine itself. Given that protein-bound Cbl is accessible to small molecules, it is likely that enzyme-bound and free intracellular Cbl(II) molecules are rapidly oxidized to inactive Cbl(III) upon exposure to peroxynitrite or (.)NO(2).
authors.affiliation: Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, USA.
authors.forename: Riya
authors.initials: R
authors.lastname: Mukherjee
authors.forename: Nicola E
authors.initials: NE
authors.lastname: Brasch
mesh_terms: D004789:Enzyme Activation; D006879:Hydroxocobalamin / Q000737:chemistry*; D007700:Kinetics; D015394:Molecular Structure; D009585:Nitrogen Dioxide / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000737:chemistry*; D011485:Protein Binding; D013057:Spectrum Analysis; D014805:Vitamin B 12 / Q000737:chemistry*
chemical_list: D030421:Peroxynitrous Acid; D014805:Vitamin B 12; D006879:Hydroxocobalamin; D009585:Nitrogen Dioxide