Abstract
Redox active cysteine residues including βCys93 are part of hemoglobin's “oxidation hotspot”. Irreversible oxidation of βCys93 ultimately leads to the collapse of the hemoglobin structure and release of heme. Human fetal hemoglobin (HbF), similarly to the adult hemoglobin (HbA), carries redox active γCys93 in the vicinity of the heme pocket. Site-directed mutagenesis has been used in this study to examine the impact of removal and/or addition of cysteine residues in HbF. The redox activities of the recombinant mutants were examined by determining the spontaneous autoxidation rate, the hydrogen peroxide induced ferric to ferryl oxidation rate, and irreversible oxidation of cysteine by quantitative mass spectrometry. We found that substitution of γCys93Ala resulted in oxidative instability characterized by increased oxidation rates. Moreover, the addition of a cysteine residue at α19 on the exposed surface of the α-chain altered the regular electron transfer pathway within the protein by forming an alternative oxidative site. This may also create an accessible site for di-sulfide bonding between Hb subunits. Engineering of cysteine residues at suitable locations may be useful as a tool for managing oxidation in a protein, and for Hb, a way to stave off oxidation reactions resulting in a protein structural collapse.
Highlights
Human fetal hemoglobin (HbF) is the main oxygen carrying protein present during the fetal development stages and up until six months after birth [1]
It was observed that αA19C and αA19C/γC93A, both containing additional cysteine located on the surface exposed part of the α-chain, behaved slightly differently compared to wildtype rHbF during purification
This is supported by cysteine quantification using Ellman's reagent which showed that the γC93A mutant only generated 30% of the signal measured for rHbF, while αA19C generated twice the signal observed in rHbF
Summary
Human fetal hemoglobin (HbF) is the main oxygen carrying protein present during the fetal development stages and up until six months after birth [1]. The γ-chain and the β-chain both consist of 146 amino acid residues but differ in their sequences at 39 or 40 positions, for Gγ and Aγ respectively, resulting in differences in some properties between HbA and HbF [2]. The oxygen affinity is higher for HbF than HbA This has been reported to be due to residues at positions γ1, γ5, γ43 and γ143 [3,4,5], resulting in lower binding of 2,3-diphosphoglycerate, which acts as an allosteric regulator. The solubility of HbF is higher than HbA, possibly due to position γ22 which is an aspartate instead of glutamate as in β-chain, and this property may contribute to the reported antisickling ability of HbF [7]. Mutants of the proposed residues could not directly verify this hypothesis [10]
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