ESR studies of free radicals in solution. II. Alkoxy radicals generated by hydrogen transfer in hydroxy thiols

Kertesz, Jean C; Wolf, Walter; Hayase, Henry

doi:10.1016/0022-2364(73)90230-8

Cited by 4 publications

(1 citation statement)

References 25 publications

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“…the primary radical product is the result of a one electron addition. The reported radicals most similar to our observed cysteinyl radical are (i) the thiyl radical produced after chemical oxidation of hydroxythiol with Ce 4ϩ in a continuous flow system at room temperature with g ϭ 2.01 and a hyperfine triplet with couplings of ϳ1.2 mT to the ␤-methylene protons (19) and (ii) the powder spectrum of a thianthrene thiyl radical cation with g av ϭ 2.0080 (2.0136, 2.0081, 2.0024) (26). The latter is strikingly similar to our powder spectrum, but better resolved, because no subtractions of overlapping EPR signals are needed.…”

Section: Discussionmentioning

confidence: 61%

Cysteinyl and Substrate Radical Formation in Active Site Mutant E441Q of Escherichia coli Class I Ribonucleotide Reductase

Persson

Sahlin

Sjöberg

1998

Journal of Biological Chemistry

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All classes of ribonucleotide reductase are proposed to have a common reaction mechanism involving a transient cysteine thiyl radical that initiates catalysis by abstracting the 3-hydrogen atom of the substrate nucleotide. In the class Ia ribonucleotide reductase system of Escherichia coli, we recently trapped two kinetically coupled transient radicals in a reaction involving the engineered E441Q R1 protein, wild-type R2 protein, and substrate (Persson, A. L., Eriksson, M., Katterle, B., Pö tsch, S., Sahlin, M., and Sjö berg, B.-M. (1997) J. Biol. Chem. 272, 31533-31541). Using isotopically labeled R1 protein or substrate, we now demonstrate that the early radical intermediate is a cysteinyl radical, possibly in weak magnetic interaction with the diiron site of protein R2, and that the second radical intermediate is a carbon-centered substrate radical with hyperfine coupling to two almost identical protons. This is the first report of a cysteinyl free radical in ribonucleotide reductase that is a kinetically coupled precursor of an identified substrate radical. We suggest that the cysteinyl radical is localized to the active site residue, Cys 439 , which is conserved in all classes of ribonucleotide reductase, and which, in the three-dimensional structure of protein R1, is positioned to abstract the 3-hydrogen atom of the substrate. We also suggest that the substrate radical is localized to the 3-position of the ribose moiety, the first substrate radical intermediate in the postulated reaction mechanism.De novo synthesis of deoxyribonucleotides from ribonucleotides is catalyzed by the enzyme ribonucleotide reductase (RNR).1 All currently known classes of RNR are considered to have a common reaction mechanism based on radical chemistry (1). The RNR classes have different subunit compositions and different metal/radical cofactor requirements, but recent structural data suggest that their substrate binding domains are homologous.2 The proposed reaction mechanism includes formation of a transient thiyl radical at the active site, which, by abstracting the 3Ј-hydrogen atom of the substrate, generates a transient 3Ј substrate radical (1, 2). The formation of this 3Ј nucleotide radical may be the critical step (3) for the subsequent steps of the reaction mechanism.The aerobic class Ia RNR of Escherichia coli, is composed of two homodimeric components, protein R1 and protein R2, of known three-dimensional structures (4, 5). Protein R1 is the substrate binding component and Cys 439 at the active site is postulated to harbor the transient thiyl radical. The cysteinyl radical is thought to be generated by long range radical transfer to the R2 component, which harbors a stable oxidized tyrosyl radical in close proximity to a diiron-oxo site. The radical transfer presumably occurs via a specific route involving at least 6 residues in R2 ( ). These residues have been conserved in class I RNRs (1), which are found in all studied eukaryotes (except Euglena), and in some eubacteria and archea (6).The in vitro k cat for the aerob...

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Section: Discussionmentioning

confidence: 61%

Cysteinyl and Substrate Radical Formation in Active Site Mutant E441Q of Escherichia coli Class I Ribonucleotide Reductase

Persson

Sahlin

Sjöberg

1998

Journal of Biological Chemistry

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EPR study of β‐methylene proton magnetic inequivalence in monomeric addition radicals derived from mono‐ and 1,1′‐disubstituted ethylenes with chiral and non‐chiral substituents

Smith

Donovan

Mader

et al. 1995

Magnetic Reson in Chemistry

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Using rapid-mixing, continuous-flow TiCI,-based techniques at 25 "C monomeric addition radicals of general formula 'CX(Y)CH,C*HRIRZ, where R' and RZ are different groups, neither being H, 90 that C* is a chiral center, were characterized by electron paramagnetic resonance. These species were generated by adding 'CH(CH,)OH, 'CH(CH,)OCH,CH, , 'CH(CH,)OC(O)H, 'CH(CH,)OC(O)CH, and 'kHCHzOCHzCHzO radicals in turn to each of the monomers, of general structure CX(Y)=CH, , acrylonitrile, acrylic acid, tert-butyl acrylate, butyl acrylate, methacrylonitrile, methacrylic acid, itaconic acid and but-3-ene-1,2,3-tricarboxylic acid. Similar reactions of carbon-centered radicals with vinyl fluoride and 1,l-difluoroethylene did not yield detectable levels of the expected addition radicals. In every addition radical observed, the splittings of the B-CH, protoy were found to be unequal and the size of this inequivalence varied qualitatively with the bulkiness of X, Y, R' and RZ. Similar measurements on monomeric addition radicals of general formula 'CX(Y)CH,Z, where Z is achiral, generated in turn from the same monomers by adding 'OH, 'NH, and 'CHOCH,OCH,d, showed no evidence for the fl-CH, proton splitting to be unequal. These results demonstrate the importance of an awareness of chirality effects in interpreting EPR spectra of monomeric and polymeric addition radicals of mono-and 1,l'-disubstituted etbylenes.

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Computer programs for ESR spectral analysis

Kertesz

Wolf

1974

Computer Programs in Biomedicine

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ESR studies of free radicals in solution. II. Alkoxy radicals generated by hydrogen transfer in hydroxy thiols

Cited by 4 publications

References 25 publications

Cysteinyl and Substrate Radical Formation in Active Site Mutant E441Q of Escherichia coli Class I Ribonucleotide Reductase

Cysteinyl and Substrate Radical Formation in Active Site Mutant E441Q of Escherichia coli Class I Ribonucleotide Reductase

EPR study of β‐methylene proton magnetic inequivalence in monomeric addition radicals derived from mono‐ and 1,1′‐disubstituted ethylenes with chiral and non‐chiral substituents

Computer programs for ESR spectral analysis

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