Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket
The reaction of hydrogen peroxide with a number of variants of sperm-whale myoglobin in which the distal pocket histidine residue (His64) had been mutated was studied with a combination of stopped-flow spectroscopy and freeze-quench EPR. The rate of the initial bimolecular reaction with hydrogen per...
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Portland Press Ltd.
1997
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crportlandpress:10.1042/bj3260109 2023-11-12T04:26:49+01:00 Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket BRITTAIN, Thomas BAKER, Adam R. BUTLER, Clive S. LITTLE, Richard H. LOWE, David J. GREENWOOD, Colin WATMOUGH, Nicholas J. 1997 http://dx.doi.org/10.1042/bj3260109 https://portlandpress.com/biochemj/article-pdf/326/1/109/624291/bj3260109.pdf en eng Portland Press Ltd. Biochemical Journal volume 326, issue 1, page 109-115 ISSN 0264-6021 1470-8728 Cell Biology Molecular Biology Biochemistry journal-article 1997 crportlandpress https://doi.org/10.1042/bj3260109 2023-10-26T21:32:45Z The reaction of hydrogen peroxide with a number of variants of sperm-whale myoglobin in which the distal pocket histidine residue (His64) had been mutated was studied with a combination of stopped-flow spectroscopy and freeze-quench EPR. The rate of the initial bimolecular reaction with hydrogen peroxide in all the proteins studied was found to depend on the polarity of the amino acid side chain at position 64. In wild-type myoglobin there were no significant optical changes subsequent to this reaction, suggesting the rapid formation of the well-characterized oxyferryl species. This conclusion was supported by freeze-quench EPR data, which were consistent with the pattern of reactivity previously reported [King and Winfield (1963) J. Biol. Chem. 238, 1520–1528]. In those myoglobins bearing a mutation at position 64, the initial bimolecular reaction with hydrogen peroxide yielded an intermediate species that subsequently decayed via a second hydrogen peroxide-dependent step leading to modification or destruction of the haem. In the mutant His64 → Gln the calculated electronic absorption spectrum of the intermediate was not that of an oxyferryl species but seemed to be that of a low-spin ferric haem. Freeze-quench EPR studies of this mutant and the apolar mutant (His64 → Val) revealed the accumulation of a novel intermediate after the first hydrogen peroxide-dependent reaction. The unusual EPR characteristics of this species are provisionally assigned to a low-spin ferric haem with bound peroxide as the distal ligand. These results are interpreted in terms of a reaction scheme in which the polarity of the distal pocket governs the rate of binding of hydrogen peroxide to the haem iron and the residue at position 64 governs both the rate of heterolytic oxygen scission and the stability of the oxyferryl product. Article in Journal/Newspaper Sperm whale Portland Press (via Crossref) Biochemical Journal 326 1 109 115 |
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Open Polar |
collection |
Portland Press (via Crossref) |
op_collection_id |
crportlandpress |
language |
English |
topic |
Cell Biology Molecular Biology Biochemistry |
spellingShingle |
Cell Biology Molecular Biology Biochemistry BRITTAIN, Thomas BAKER, Adam R. BUTLER, Clive S. LITTLE, Richard H. LOWE, David J. GREENWOOD, Colin WATMOUGH, Nicholas J. Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket |
topic_facet |
Cell Biology Molecular Biology Biochemistry |
description |
The reaction of hydrogen peroxide with a number of variants of sperm-whale myoglobin in which the distal pocket histidine residue (His64) had been mutated was studied with a combination of stopped-flow spectroscopy and freeze-quench EPR. The rate of the initial bimolecular reaction with hydrogen peroxide in all the proteins studied was found to depend on the polarity of the amino acid side chain at position 64. In wild-type myoglobin there were no significant optical changes subsequent to this reaction, suggesting the rapid formation of the well-characterized oxyferryl species. This conclusion was supported by freeze-quench EPR data, which were consistent with the pattern of reactivity previously reported [King and Winfield (1963) J. Biol. Chem. 238, 1520–1528]. In those myoglobins bearing a mutation at position 64, the initial bimolecular reaction with hydrogen peroxide yielded an intermediate species that subsequently decayed via a second hydrogen peroxide-dependent step leading to modification or destruction of the haem. In the mutant His64 → Gln the calculated electronic absorption spectrum of the intermediate was not that of an oxyferryl species but seemed to be that of a low-spin ferric haem. Freeze-quench EPR studies of this mutant and the apolar mutant (His64 → Val) revealed the accumulation of a novel intermediate after the first hydrogen peroxide-dependent reaction. The unusual EPR characteristics of this species are provisionally assigned to a low-spin ferric haem with bound peroxide as the distal ligand. These results are interpreted in terms of a reaction scheme in which the polarity of the distal pocket governs the rate of binding of hydrogen peroxide to the haem iron and the residue at position 64 governs both the rate of heterolytic oxygen scission and the stability of the oxyferryl product. |
format |
Article in Journal/Newspaper |
author |
BRITTAIN, Thomas BAKER, Adam R. BUTLER, Clive S. LITTLE, Richard H. LOWE, David J. GREENWOOD, Colin WATMOUGH, Nicholas J. |
author_facet |
BRITTAIN, Thomas BAKER, Adam R. BUTLER, Clive S. LITTLE, Richard H. LOWE, David J. GREENWOOD, Colin WATMOUGH, Nicholas J. |
author_sort |
BRITTAIN, Thomas |
title |
Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket |
title_short |
Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket |
title_full |
Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket |
title_fullStr |
Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket |
title_full_unstemmed |
Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket |
title_sort |
reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket |
publisher |
Portland Press Ltd. |
publishDate |
1997 |
url |
http://dx.doi.org/10.1042/bj3260109 https://portlandpress.com/biochemj/article-pdf/326/1/109/624291/bj3260109.pdf |
genre |
Sperm whale |
genre_facet |
Sperm whale |
op_source |
Biochemical Journal volume 326, issue 1, page 109-115 ISSN 0264-6021 1470-8728 |
op_doi |
https://doi.org/10.1042/bj3260109 |
container_title |
Biochemical Journal |
container_volume |
326 |
container_issue |
1 |
container_start_page |
109 |
op_container_end_page |
115 |
_version_ |
1782340655415033856 |