Abstract
The highly toxic oxidative transformation of hemoglobin (Hb) to the ferryl state (HbFe4+) is known to occur in both in vitro and in vivo settings. We recently constructed oxidatively stable human Hbs, based on the Hb Providence (βK82D) mutation in sickle cell Hb (βE6V/βK82D) and in a recombinant crosslinked Hb (rHb0.1/βK82D). Using High Resolution Accurate Mass (HRAM) mass spectrometry, we first quantified the degree of irreversible oxidation of βCys93 in these proteins, induced by hydrogen peroxide (H2O2), and compared it to their respective controls (HbA and HbS). Both Hbs containing the βK82D 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 βK82D containing Hbs on vascular endothelial redox homeostasis and energy metabolism. Incubation of the mutants carrying βK82D with endothelial cells resulted in altered bioenergetic function, by improving basal cellular glycolysis and glycolytic capacity. Treatment of cells with Hb variants containing βK82D resulted in lower heme oxygenase-1 and ferritin expressions, compared to native Hbs. We conclude that the presence of βK82D confers oxidative stability to Hb and adds significant resistance to oxidative toxicity. Therefore, we propose that βK82D is a potential gene-editing target in the treatment of sickle cell disease and in the design of safe and effective oxygen therapeutics.
Highlights
Heme iron oxidation can have serious biological consequences as it impacts the ability of Hb to deliver oxygen, which can jeopardize its safe use in transfusion medicine [1]
We found a strong correlation between the suppression of βCys93 oxidation in Hb proteins containing βK82D mutation and the recovery of endothelial glycolytic capacity, along with diminished heme oxygenase-1 (HO-1) expression, which is caused by the toxic release of heme
Mutant Hbs are described as “Experiments in Nature”, as they provide an experimental platform to investigate why some mutants evolutionarily evolved into oxidatively stable molecules, while others develop into circulatory disorders [26,27]
Summary
Heme iron oxidation can have serious biological consequences as it impacts the ability of Hb to deliver oxygen, which can jeopardize its safe use in transfusion medicine [1]. When RBCs are stored or pathogen inactivated for transfusion purposes, the function of these antioxidant mechanisms can be compromised [4]. RBC genetic disorders and hemoglobinopathies accelerate Hb oxidation, which often leads to premature hemolysis and heme loss [5]. As a result, understanding the underlying mechanisms of Hb oxidation is critical for designing methods aimed at potentially reducing or preventing its complications
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