Kinetics and mechanistic aspects of superoxide radical-mediated transformation of ascorbate

Ascorbate (Asc) has long been known to be an antioxidant against reactive oxygen species (ROS) for cytotoxicity and human diseases. However, its reported reactivity with superoxide radical (O-2(center dot-)), an important ROS involved in aerobic metabolism system, exhibits great discrepancy; its mechanistic mode of actions on the molecular level is also largely debatable. Here, we investigated the reaction of Asc and O-2(center dot-) by a combination of experimental and theoretical approaches. The rate constant measured by a competitive technique method was determined to be 3.3 x 10(4) M-1 s(-1) under physiological condition. Mechanistically, our theoretical result indicated that O-2(center dot-) abstracts a hydrogen atom on the enolic hydroxyl group forming ascorbyl radical (Asc(center dot)), which is unreactive with O-2(center dot-) or Asc center dot itself. However, with different protonation state of Asc(center dot) (i.e., AscH(center dot)) in acidic environment, its reactivity was demonstrated to be significantly altered. AscH(center dot) can react with O-2(center dot-) via hydrogen atom abstraction and single electron transfer and/or undergo self-disproportionation. These complementary results provided fundamental principles for the ecotoxicological function and environmental potentials of Asc against ROS.