SUPEROXIDE AND HYDROGEN PEROXIDE-DEPENDENT INHIBITION OF IRON REGULATORY PROTEIN-ACTIVITY - A PROTECTIVE STRATAGEM AGAINST OXIDATIVE INJURY

Cellular iron homeostasis is regulated by the cytoplasmic iron regulat ory protein (IRP), which binds to iron-responsive elements (IRE) of mR NAs, modulating iron uptake and sequestration, respectively. When iron is scarce, IRP binds to IRE and coordinately increases the synthesis of transferrin receptor and decreases that of ferritin, thus providing the cell with readily available free iron. When iron is in excess, IR P does not bind and iron sequestration prevails over iron uptake. We h ave found that incubation of rat liver lysates with xanthine oxidase ( XO), which generates superoxide (O-2(.-)) and hydrogen peroxide (H2O2) , caused a remarkable but reversible inhibition of IRP activity, as th e formation of IRE-IRP decreased by 70-80% but returned to baseline va lues upon exposure to a reducing agent like 2-mercaptoethanol. IRP inh ibition was prevented by separate or simultaneous addition of superoxi de dismutase and catalase, showing that both O-2(.-). and H2O2 were in volved. By contrast, iron chelators and hydroxyl radical scavengers di d not impede the inhibition of IRP, suggesting that O-2(.-) and H2O2 a cted independently of fi ee iron sources. Ferritin enhanced IRP inhibi tion, but this process involved tightly bound iron centers that shunte d reducing equivalents from XO and returned them to oxygen, thus incre asing the formation of O-2(.-). In agreement with the exclusive role o f O-2(.-) and H2O2, XO also inhibited recombinant human IRP in the abs ence of iron. These results demonstrate that O2(.-). and H2O2 can dire ctly but reversibly down-regulate the RNA-binding activity of IRP, cau sing transient decrease of free iron that otherwise would convert them into more potent oxidants such as hydroxyl radicals or equally aggres sive iron-peroxo complexes. This establishes a novel protective strata gem against oxidative injury under pathophysiologic conditions charact erized by the excessive generation of O2(.-) and H2O2.