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.