Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin

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.

Site-Specific Introduction of Negative Charges on the Protein Surface for Improving Global Functions of Recombinant Fetal Hemoglobin

“…Site-directed mutagenesis of the α-subunit gene, previously inserted into a pETDuet-1 plasmid containing both the α- and γ-subunit genes, was performed as described before (Ratanasopa et al, 2016 ; Kettisen et al, 2018 ). The locations of the mutations are displayed in Figure 1 .…”

“…Primers were ordered from Integrated DNA Technologies (Germany). Sequence confirmation and transformation of mutated plasmids into Escherichia coli BL21 (DE3), as well as expression and purification of wtHbF and all three mutants, were performed as previously described (Kettisen et al, 2018 ), with a few modifications. In short, 600 ml Terrific Broth cultures in 2 L Erlenmeyer shake flasks, containing 0.3 mM δ-aminolevulinic acid (Sigma-Aldrich), were induced at OD 0.1 with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Saveen & Werner) and cultivated at 30°C overnight.…”

“…The gel was fixed in 12% trichloroacetic acid for 30 min and then stained with Coomassie blue to visualize the protein bands. Autoxidation rates of 10 μM Hb-O 2 to Hb (Fe 3+ ) were examined by recording the spectral change every 30 min at 20°C for 24 h; and the heme loss were monitored at 37°C for 8 h with the heme scavenger apomyoglobin H64Y/V67F (apoMb) (Hargrove et al, 1994 ); according to procedures described previously (Kettisen et al, 2018 ). A component analysis in the spectrum range 350–700 nm was used to obtain the heme loss rates (Singh et al, 2020 ).…”

“…Compared to adult hemoglobin (HbA, α 2 β 2 ), HbF has several differentiating qualities, such as a higher alkali denaturation resistance (Perutz, 1974 ), higher solubility (Frier and Perutz, 1977 ), lower tetramer-dimer dissociation constant (Yagami et al, 2002 ), and higher affinity for oxygen in the presence of 2,3-diphosphoglycerate (Bauer et al, 1968 ; Bunn and Briehl, 1970 ). In previous work, we reported a mutant of HbF, which showed decreased oxidative reactivity compared to wild-type HbF (wtHbF) (Kettisen et al, 2018 ). This mutant is part of an effort to examine rational site-directed mutagenesis to mitigate adverse oxidative reactivity of future Hb-based products.…”

Reactive Oxygen Species and Their Involvement in Red Blood Cell Damage in Chronic Kidney Disease

“…The disturbance of the redox balance is associated with the increase in ROS production and a decrease in antioxidant capacity. In turn, irreversible oxidation of the residue of the cysteine β Cys93 in the globin chain may lead to disintegration of the structure of Hb and, consequently, to the release of heme, which also catalyzes free radical reactions [ 138 ]. There is a decrease in the activity of antioxidant enzymes [ 119 , 121 , 123 ].…”