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Biochemistry. 2008 Sep 30;47(39):10440-8. doi: 10.1021/bi800771k. Epub 2008 Sep 4.

Identification of protein radicals formed in the human neuroglobin-H2O2 reaction using immuno-spin trapping and mass spectrometry.

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1
Laboratory of Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA. lardinois.olivier@gmail.com

Abstract

Neuroglobin (Ngb) is a recently discovered protein that shows only minor sequence similarity with myoglobin and hemoglobin but conforms to the typical 3-over-3 alpha-helical fold characteristic of vertebrate globins. An intriguing feature of Ngb is its heme hexacoordination in the absence of external ligands, observed both in the ferrous and in the ferric (met) forms. In Ngb, the imidazole of a histidine residue (His-64) in the distal position, above the heme plane, provides the sixth coordination bond. In this work, a valine residue was introduced at position 64 (H64V variant) to clarify the possible role(s) of the distal residue in protecting the heme iron of Ngb from attack by strong oxidants. SDS-PAGE analyses revealed that the oxidation of the H64V variant of metNgb by H 2O 2 resulted in the formation of dimeric and trimeric products in contrast to the native protein. Dityrosine cross-links were shown by their fluorescence to be present in the oligomeric products. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was included in the reaction mixture, nitrone adducts were detected by immuno-spin trapping. The specific location of the DMPO adducts on the H64V variant protein was determined by a mass spectrometry method that combines off-line immuno-spin trapping and chromatographic procedures. This method revealed Tyr-88 to be the site of modification by DMPO. The presence of His-64 in the wild-type protein results in the nearly complete loss of detectable radical adducts. Together, the data support the argument that wild-type Ngb is protected from attack by H 2O 2 by the coordinated distal His.

PMID:
18767815
PMCID:
PMC2685255
DOI:
10.1021/bi800771k
[Indexed for MEDLINE]
Free PMC Article
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