The Providence Mutation (Beta K82d) In Human Hemoglobin Substantially Reduces Beta Cysteine 93 Oxidation And Oxidative Stress In Endothelial Cells

The highly toxic oxidative transformation of hemoglobin (Hb) to the ferryl state (HbFe(4+)) is known to occur in both in vitro and in vivo settings. We recently constructed oxidatively stable human Hbs, based on the Hb Providence (beta K82D) mutation in sickle cell Hb (beta E6V/beta K82D) and in a recombinant crosslinked Hb (rHb0.1/beta K82D). Using High Resolution Accurate Mass (HRAM) mass spectrometry, we first quantified the degree of irreversible oxidation of beta Cys93 in these proteins, induced by hydrogen peroxide (H2O2), and compared it to their respective controls (HbA and HbS). Both Hbs containing the beta K82D mutation showed considerably less cysteic acid formation, a byproduct of cysteine irreversible oxidation. Next, we performed a novel study aimed at exploring the impact of introducing beta K82D containing Hbs on vascular endothelial redox homeostasis and energy metabolism. Incubation of the mutants carrying beta K82D with endothelial cells resulted in altered bioenergetic function, by improving basal cellular glycolysis and glycolytic capacity. Treatment of cells with Hb variants containing beta K82D resulted in lower heme oxygenase-1 and ferritin expressions, compared to native Hbs. We conclude that the presence of beta K82D confers oxidative stability to Hb and adds significant resistance to oxidative toxicity. Therefore, we propose that beta K82D is a potential gene-editing target in the treatment of sickle cell disease and in the design of safe and effective oxygen therapeutics.