Detection and identification of the oxidizing species generated from the physiologically important Fenton-like reaction of iron(II)-citrate with hydrogen peroxide

Abstract

The Fenton-like reaction of iron(II)-citrate with hydrogen peroxide is physiologically important because it is associated with the oxidative stress and pathological processes induced by the redox-active iron pool in vivo. However, the oxidizing species generated from this reaction at neutral pH has not been convincingly identified because two extremely unstable and hard-to-differentiate species, the hydroxyl radical (^•OH) and iron(IV) (ferryl) species, can be produced. Identifying this species is essential for understanding the reaction mechanism. Although there were few data that reported the detection of ^•OH from this reaction by using the EPR and fluorescence techniques, most of these data were obtained without the necessary assessment with a ^•OH scavenger. Furthermore, these two techniques may not be able to differentiate the ^•OH and iron(IV) species. Thus, these reported data cannot lead to a convincing conclusion that the ^•OH, not the iron(IV) species, was generated. Therefore, in the study reported herein, we carried out systematic investigations first by using the EPR and fluorescence techniques combined with a ^•OH scavenger to detect the oxidizing species generated from this Fenton-like reaction. Then we utilized NMR spectroscopy and for the first time obtained convincing evidence to demonstrate that this oxidizing species is the ^•OH rather than iron(IV) species. We also determined the second-order rate constant of the reaction, 3.6 × 10³ M^-1s^-1 (pH7.0, 25 °C), by using the stopped-flow spectrophotometry. On the basis of these findings, a scheme is proposed for the mechanism of this physiologically important Fenton-like reaction.

Keywords: Fenton-like reaction; Hydroxyl radical; Iron(II)-Citrate complex; Iron(IV) (ferryl) species; Oxidative stress; Redox-active iron pool.