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Haptoglobin binding stabilizes Hemoglobin Ferryl Iron and the Globin Radical on Tyrosine β145


Cooper, Chris E; Schaer, Dominik J; Buehler, Paul W; Wilson, Michael T; Reeder, Brandon J; Silkstone, Gary; Svistunenko, Dimitri A; Bulow, Leif; Alayash, Abdu I (2013). Haptoglobin binding stabilizes Hemoglobin Ferryl Iron and the Globin Radical on Tyrosine β145. Antioxidants & Redox Signaling, 18(17):2264-2273.

Abstract

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 sitedirected 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 Hbcatalyzed 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.

Abstract

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 sitedirected 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 Hbcatalyzed 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.

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Contributors:School of Biological Sciences, University of Essex
Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Clinic and Policlinic for Internal Medicine
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2013
Deposited On:07 Feb 2013 08:31
Last Modified:04 Aug 2017 01:49
Publisher:Mary Ann Liebert
ISSN:1523-0864
Additional Information:This is a copy of an article published in the Antioxidants & Redox Signaling © 2012 Mary Ann Liebert, Inc.; Antioxidants & Redox Signaling is available online at: http://www.liebertonline.com.
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1089/ars.2012.4547
PubMed ID:22702311

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