Hemin Recapitulates the Labile Iron Pool in the Cellular Fenton Reaction with DNA.

The iron-Fenton reaction in biology is influenced by diverse cellular molecules that coordinate with redox-active ferrous ion. Contemporary research proposed that hydroxyl radical or a ferryl species (HO•/Fe=O2+) are the primary oxidants; however, this was challenged by the observation that physiological bicarbonate redirects the reaction to form carbonate radical anion (CO3•-). Questions remained regarding the roles of O2 concentration, ascorbate, and iron speciation in CO3•- formation. Accordingly, in cellulo studies were conducted under physiological O2 (∼25 μM) with ascorbate replenishment to monitor bicarbonate-dependent telomeric DNA damage. Under these conditions,. physiological bicarbonate (25 mM) yielded 2'-deoxyguanosine-specific oxidation consistent with CO3•- formation at a ratio exceeding 80:1 relative to HO•/Fe=O2+. In parallel in vitro experiments using a plasmid nicking assay, the cellular low molecular weight (LMW) ultrafiltrate was used as the source of iron and its endogenous coordination partners; under physiological O2, bicarbonate, and 500 nM H2O2 (the concentration required to produce detectable signal), the DNA damage profile was consistent with exclusive CO3•- formation, mirroring the cell culture result. A panel of iron complexes approximating the intracellular labile iron pool (5 μM) was examined: hexaaquo-ferrous ion, ferrous citrate, ferrous α-ketoglutarate, ferrous pyrophosphate, ferrous glutathione, and hemin. Of these, only hemin reproduced the bicarbonate-dependent CO3•- damage profile observed in cells with 100 nM H2O2 and 25 mM bicarbonate present. This finding was corroborated using a defined biomimetic metabolome in which hemin, ferrous ion, or their combination was tested; hemin consistently supported CO3•- as the dominant oxidant. Roles for the Udenfriend reaction (Fe(II), O2, and reductant) and superoxide dismutase were also studied. Collectively, these results identify heme iron as a likely candidate to drive CO3•- formation via the bicarbonate iron-Fenton reaction to damage dG in DNA during endogenous oxidative stress.