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Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Department of Biochemistry and Biophysics.
Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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2002 (English)In: J Biol Inorg Chem, ISSN 0949-8257, Vol. 7, no 1-2, 74-82 p.Article in journal (Other academic) Published
Abstract [en]

Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.

Place, publisher, year, edition, pages
2002. Vol. 7, no 1-2, 74-82 p.
Keyword [en]
Cysteine/chemistry, Electron Spin Resonance Spectroscopy/methods, Escherichia coli/*enzymology, Glycine/chemistry, Iron/*chemistry, Oxygen/*chemistry, Peroxides/*chemistry, Point Mutation, Ribonucleotide Reductases/*chemistry/genetics, Tryptophan/chemistry, Tyrosine/chemistry
Identifiers
URN: urn:nbn:se:su:diva-20353PubMedID: 11862543OAI: oai:DiVA.org:su-20353DiVA: diva2:186879
Available from: 2007-03-10 Created: 2007-03-10 Last updated: 2011-01-13Bibliographically approved

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Sahlin, MargaretaSjöberg, Britt-MarieGräslund, Astrid
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