Haptoglobin binding stabilizes hemoglobin ferryl iron and the globin radical on tyrosine β145

Abstract

Aim: Hemoglobin (Hb) becomes toxic when released from the erythrocyte. The acute phase protein haptoglobin (Hp) binds avidly to Hb and decreases oxidative damage to Hb itself and to the surrounding proteins and lipids. However, the molecular mechanism underpinning Hp protection is to date unclear. The aim of this study was to use electron paramagnetic resonance (EPR) spectroscopy, stopped flow optical spectrophotometry, and site-directed mutagenesis to explore the mechanism and specifically the role of specific tyrosine residues in this protection.

Results: Following peroxide challenge Hb produces reactive oxidative intermediates in the form of ferryl heme and globin free radicals. Hp binding increases the steady state level of ferryl formation during Hb-catalyzed lipid peroxidation, while at the same time dramatically inhibiting the overall reaction rate. This enhanced ferryl stability is also seen in the absence of lipids and in the presence of external reductants. Hp binding is not accompanied by a decrease in the pK of ferryl protonation; the protonated ferryl species still forms, but is intrinsically less reactive. Ferryl stabilization is accompanied by a significant increase in the concentration of the peroxide-induced tyrosine free radical. EPR spectral parameters and mutagenesis studies suggest that this radical is located on tyrosine 145, the penultimate C-terminal amino acid on the beta Hb subunit.

Innovation: Hp binding decreases both the ferryl iron and free radical reactivity of Hb.

Conclusion: Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging.

FIG. 1.

Peroxidase cycle of Hb. The two-electron oxidant hydrogen peroxide reacts with ferric hemoglobin to form (initially) ferryl heme and a free radical. Both these species can be reduced by external reductants, such as ascorbate, reforming ferric hemoglobin. In the absence of such a reductant, the free radical can migrate within the protein, forming globin free radicals detectable on tryrosine residues. The ferryl heme itself can autoreduce to reform the ferric protein. Uncontrolled oxidative chemistry at the heme and the free radical sites can result in oxidative modification to hemoglobin itself or nearby biomolecules. DHA, dehydroascorbate; Fe^III, Ferric(met) hemoglobin; Hb, hemoglobin; Fe^IV=O, ferryl hemoglobin; Tyr, deprotonated tyrosine radical. “?” indicates uncertainties about precise routes of electron transfer and the ultimate fate of the electron hole caused by reduction of the primary cation radical.

FIG. 2.

Optical spectra of Hb during lipid peroxidation. Ferryl Hb was incubated with liposomes in the absence (control) and presence of +HSA and +Hp. Conditions: [Hb]=4 μM, [HSA]=1.2 μM, [Hp]=4.8 μM, 20 mM sodium phosphate, pH=7.4, T=22°C. (a) Top: spectra taken at 5 min after catalase addition; bottom: standard Hb spectra for comparison. The percentage of ferryl remaining after 5 min is also indicated. (b) Time course of optical changes in the Soret wavelength region. Analysis of the full wavelength spectra shows that the first 30 min are consistent with peroxide-induced conversion of ferric heme to ferryl followed by a slower decay back to ferric after the peroxide is removed by catalase. The decrease in absorbance after 30 min in the absence of haptoglobin corresponded to a general bleaching of the heme spectra. (c) UV absorbance changes corresponding to conjugated diene formation. Hp, haptoglobin; HSA, human serum albumin.

FIG. 3.

Hp inhibition of HPODE oxidation. (a) Ferric Hb or Hb:Hp complex (both 5 μM heme) was reacted with HPODE (40 μM) in 25 mM sodium phosphate (pH 7.4). An increase in absorbance at 425–408 nm indicates conversion of ferric to ferryl Hb. Mean±SD, n=3. (b) The effect of HSA and Hp on the maximum absorbance achieved at 425–408 nm (normalized to 1 for the control); Hp point offset for clarity. HPODE, 13-S 9-cis, 11-trans octadecadienoic acid; SD, standard deviation.

FIG. 4.

Effect of pH on the ferryl Hb:Hp complex. (a) The pH dependence of the initial spectrum (1.2 ms) following mixing of 6 μM Hb and 8 μM Hp at pH 8.6 with buffers at lower pH. Final pH varied from 8.6 to 2.2. (b) Nonlinear regression fit of absorbance change following mixing for Hb (open circles) and Hb:Hp complex (filled circles). The two data sets gave no difference in the pK for the initial absorbance change: 4.27±0.07 for the control and 4.27±0.05 in the presence of Hp. The fit illustrated is to the combined data sets with pK of 4.27±0.04. (c) Changes in absorbance with time after pH jumping from 8.6 to 2.85. Spectra are shown from 0.1 to 2 s at 0.1 s intervals. (d) Time courses at different pH values for Hb (black lines) and Hb:Hp complex (gray lines). The pH jumps were 4.15, 3.55, 2.85, and 2.55.

FIG. 5.

Ferryl Hb reduction to metHb. Ferryl Hb or ferryl Hb:Hp complex (both 10 μM heme) in 25 mM sodium phosphate buffer. (a) Effect of pH on the autoreduction rate. Data fitted to pK of 4.27; maximum rate is indicated in inset. (b) Effect of phenol on the reduction rate at pH 7.4. Inset shows second-order rate constant calculated from the slope of the curve. metHb, methemoglobin.

FIG. 6.

Effect of Hp on the formation of heme to protein cross-linked Hb. MetHb (80 μM) in the absence or presence of Hp (80 μM) was reacted with hydrogen peroxide at pH 5.0 (0.1 M sodium acetate containing 100 μM DTPA). The reaction was allowed to stand for 2 h before analysis by HPLC. Note that at high peroxide concentrations the heme is damaged, leading to a decrease in the ability to detect heme to protein cross-linked Hb rather than a decrease in the extent of its formation.

FIG. 7.

EPR spectra following peroxide addition to Hb. The effect of Hp (80 μM) on the low-temperature EPR spectra following hydrogen peroxide (80 μM) addition to metHb (80 μM) (pH 7.4) and 20 mM sodium phosphate. The spectra were recorded at 10 K. Inset table shows calculated ferryl and free radical concentrations (mean±SD, n=3). *Significant difference (p<0.05) in two-way ANOVA for both time and presence/absence of Hp. EPR, electron paramagnetic resonance.

FIG. 8.

EPR parameters for the tyrosine radical in the Hb:Hp complex. Simulation of the Hp:Hb radical. (A) Experimental spectrum of 80 μM Hp:Hb complex reacting with 80 μM H₂O₂, frozen 150 s after mixing. (B) Simulated spectrum obtained by our algorithm (44) for spin density on tyrosine atom: C1 ρ_C1= 0.410 and ring rotation angle θ=73°. Note the simulation cannot distinguish between θ=73° and θ=47 (120–73)°.

FIG. 9.

Effect of mutation at βTyr145 on tyrosine radical formation. Free radical formation in the wild type and Tyr145Phe mutant. Experimental conditions as per Figure 7 but with 60 μM peroxide added to 60 μM metHb. WT, wild type.