Abstract
Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H2O2) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H2O2 molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H2O2 at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized βCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.
Highlights
HbS oxidation is recognized as an important element in the pathophysiology of sickle cell disease
We found that the ferryl form of HbS promotes a significant impediment of mitochondrial respiration and oxidative stress in lung cells
HbO2 readily undergoes a source of reactive oxygen species (ROS), namely oxidation of its heme iron that become anionic superoxide (O2. ), which subsequently dismutates to H2O2
Summary
HbS oxidation is recognized as an important element in the pathophysiology of sickle cell disease. The heme-TLR4 complex activates NF-B and triggers vaso-occlusion through Weibel-Palade body degranulation and adhesion molecule expression [11] Based on these observations, it is not surprising that heme (derived from sickle RBC hemolysis) has recently been described as a damage-associated molecular pattern molecule driving inflammation [11, 12]. Ferryl Hb can spontaneously autoreduce back to the ferric form completing a catalytic cycle (see Scheme 1) Both the ferryl heme and the protein cation radical induce a wide variety of oxidative reactions affecting both the protein itself and nearby molecules due to their high midpoint redox potentials (E1° /2 ϳ1.0 V) [13].
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