Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin

Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation after hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally nonequivalent. We have investigated the functional and redox properties of Hb–Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9 μM−1 s−1, irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E1/2) of the ferric/ferrous pair of each Hb–Hp complex are similar, varying from +54 to +59 mV (vs NHE), and are essentially the same as reported by us previously for Hb–Hp complexes prepared from unfractionated Hp. All Hb–Hp complexes generate similar high amounts of ferryl Hb after exposure to hydrogen peroxide. Electron paramagnetic resonance data indicate that the yields of protein-based radicals during this process are approximately 4 to 5% and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to one another with respect to Hb binding and associated stability and redox properties and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease

Alpha-hemoglobin stabilizing protein (AHSP) markedly decreases the redox potential and reactivity of alpha subunits of human HbA with hydrogen peroxide

Background: AHSP modifies redox properties of bound α subunits. Results: Isolated hemoglobin subunits exhibit significantly different redox properties compared to HbA. A significant decrease in the reduction potential of α subunits bound to AHSP results in preferential binding of ferric α. Conclusion: AHSP:α subunit complexes do not participate in ferric-ferryl heme redox cycling. Significance: AHSP binding to α subunits inhibits subunit pseudoperoxidase activity

Post-translational Transformation of Methionine to Aspartate Is Catalyzed by Heme Iron and Driven by Peroxide: A NOVEL SUBUNIT-SPECIFIC MECHANISM IN HEMOGLOBIN

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in ? subunits of human fetal Hb Toms River (?67(E11)Val ? Met) and ? subunits of adult Hb (HbA) Bristol-Alesha (?67(E11)Val ? Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met ? Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H218O2, we verified incorporation of 18O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the ?Met(E11) position in the corresponding HbA Evans (?62(E11)Val ? Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in ? subunits but not in ? subunits. This novel post-translational modification highlights the nonequivalence of human Hb ?, ?, and ? subunits with respect to redox reactivity and may have direct implications to ?/? hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics

Analysis of human α globin gene mutations that impair binding to the α hemoglobin stabilizing protein

Alpha hemoglobin stabilizing protein (AHSP) reversibly binds nascent α globin to maintain its native structure and facilitate its incorporation into hemoglobin A. Previous studies indicate that some naturally occurring human α globin mutations may destabilize the protein by inhibiting its interactions with AHSP. However, these mutations could also affect hemoglobin A production through AHSP-independent effects, including reduced binding to β globin. We analyzed 6 human α globin variants with altered AHSP contact surfaces. Alpha globin amino acid substitutions H103Y, H103R, F117S, and P119S impaired interactions with both AHSP and β globin. These mutations are destabilizing in biochemical assays and are associated with microcytosis and anemia in humans. By contrast, K99E and K99N α globins bind β globin normally but exhibit attenuated binding to AHSP. These mutations impair protein folding and expression in vitro and appear to be mildly destabilizing in vivo. In Escherichia coli and erythroid cells, α globin K99E stability is rescued on coexpression with AHSP mutants in which binding to the abnormal globin chain is restored. Our results better define the biochemical properties of some α globin variants and support the hypothesis that AHSP promotes α globin chain stability during human erythropoiesis

The Role of Alpha-Hemoglobin Stabilizing Protein in Redox Chemistry, Denaturation, and Hemoglobin Assembly

Hemoglobin biosynthesis in erythrocyte precursors involves several steps. The correct ratios and concentrations of normal alpha (α) and beta (β) globin proteins must be expressed; apoproteins must be folded correctly; heme must be synthesized and incorporated into these globins rapidly; and the individual α and β subunits must be rapidly and correctly assembled into heterotetramers. These events occur on a large scale in vivo, and dysregulation causes serious clinical disorders such as thalassemia syndromes. Recent work has implicated a conserved erythroid protein known as Alpha-Hemoglobin Stabilizing Protein (AHSP) as a participant in these events. Current evidence suggests that AHSP enhances α subunit stability and diminishes its participation in harmful redox chemistry. There is also evidence that AHSP facilitates one or more early-stage post-translational hemoglobin biosynthetic events. In this review, recent experimental results are discussed in light of several current models describing globin subunit folding, heme uptake, assembly, and denaturation during hemoglobin synthesis. Particular attention is devoted to molecular interactions with AHSP that relate to α chain oxidation and the ability of α chains to associate with partner β chains. Antioxid. Redox Signal. 12, 219–232