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.