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

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 a...

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Autores principales: Kettisen, Karin, Strader, Michael Brad, Wood, Francine, Alayash, Abdu I., Bülow, Leif
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126208/
https://www.ncbi.nlm.nih.gov/pubmed/30193183
http://dx.doi.org/10.1016/j.redox.2018.08.010
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author Kettisen, Karin
Strader, Michael Brad
Wood, Francine
Alayash, Abdu I.
Bülow, Leif
author_facet Kettisen, Karin
Strader, Michael Brad
Wood, Francine
Alayash, Abdu I.
Bülow, Leif
author_sort Kettisen, Karin
collection PubMed
description 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.
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spelling pubmed-61262082018-09-06 Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin() Kettisen, Karin Strader, Michael Brad Wood, Francine Alayash, Abdu I. Bülow, Leif Redox Biol Research Paper 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. Elsevier 2018-08-22 /pmc/articles/PMC6126208/ /pubmed/30193183 http://dx.doi.org/10.1016/j.redox.2018.08.010 Text en © 2018 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Paper
Kettisen, Karin
Strader, Michael Brad
Wood, Francine
Alayash, Abdu I.
Bülow, Leif
Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin()
title Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin()
title_full Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin()
title_fullStr Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin()
title_full_unstemmed Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin()
title_short Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin()
title_sort site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin()
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126208/
https://www.ncbi.nlm.nih.gov/pubmed/30193183
http://dx.doi.org/10.1016/j.redox.2018.08.010
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