Manganese(II)-bicarbonate-mediated catalytic activity for hydrogen peroxide dismutation and amino acid oxidation: detection of free radical intermediates.
To examine the structural identities of reactive free radicals and the mechanism of the oxidative modification of proteins, we used EPR and spin-trapping methods to investigate the oxidation of amino acids by H2O2 as well as the decomposition of H2O2 itself catalyzed by Mn(II) ions. Superoxide and hydroxyl radicals (O2-. and OH.) were trapped by a spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), in a reaction mixture containing Mn(II) and H2O2 in bicarbonate/CO2 buffer. When Hepes was used in place of bicarbonate buffer, superoxide radical was not observed, indicating the importance of bicarbonate buffer. With addition of L-leucine to a similar reaction mixture, a leucine-derived radical that replaced the DMPO-superoxide adduct was detected in the absence and presence of DMPO. Using various isotope-enriched L-leucines, we successfully identified this radical as a hydronitroxide, -OOC(R)CHNHO.. The data are consistent with the formation of a transient "caged" OH. in the inner coordination sphere of Mn(II). This caged OH. is likely to undergo an intramolecular hydrogen-atom abstraction from the Mn-bound H2O2 or amino acid. Two reaction schemes are proposed to account for the experimental results shown here and in the preceding papers.
Yim
M B
MB
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.
Berlett
B S
BS
Chock
P B
PB
Stadtman
E R
ER
D000596:Amino Acids; D001639:Bicarbonates; D002384:Catalysis; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006861:Hydrogen Peroxide; D006878:Hydroxides; D017665:Hydroxyl Radical; D007700:Kinetics; D008345:Manganese; D008954:Models, Biological; D010084:Oxidation-Reduction; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000378:metabolism
D000596:Amino Acids; D001639:Bicarbonates; D003497:Cyclic N-Oxides; D005609:Free Radicals; D006878:Hydroxides; D013113:Spin Labels; D017665:Hydroxyl Radical; D008345:Manganese; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase