Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2

Sara E. Parkin, Shuxian Chen, Brenda A. Ley, Lara Mangravite, Dale E. Edmondson, Boi Hanh Huynh, Joseph M. Bollinger, Jr.

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122. A key step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O2. As this step leaves only one of the two oxidizing equivalents of the initial diiron(II)-O2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases). In the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction [Aberg, A., Ormo, M., Nordlund, P., and Sjoberg, B. M. (1993) Biochemistry 32, 9845-9850], implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes. Here we demonstrate that O2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (namely, high concentration of the reductant ascorbate). Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent on the integrity of a hydrogen-bond network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration. These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface.

Original languageEnglish (US)
Pages (from-to)1124-1130
Number of pages7
JournalBiochemistry
Volume37
Issue number4
DOIs
StatePublished - Jan 27 1998

Fingerprint

Ribonucleotide Reductases
Electron injection
Escherichia coli
Chemical activation
Electrons
Oxygen
Injections
Reducing Agents
Proteins
Hydroxylation
Oxidation
Hydrogen bonds
Cytochrome P-450 CYP4A
Hydrogen
Biochemistry
Membrane Proteins
Substitution reactions
Iron
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Parkin, Sara E. ; Chen, Shuxian ; Ley, Brenda A. ; Mangravite, Lara ; Edmondson, Dale E. ; Huynh, Boi Hanh ; Bollinger, Jr., Joseph M. / Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2. In: Biochemistry. 1998 ; Vol. 37, No. 4. pp. 1124-1130.
@article{ca3bbf80ed5e486e9693a85a59bd0d3c,
title = "Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2",
abstract = "Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122. A key step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O2. As this step leaves only one of the two oxidizing equivalents of the initial diiron(II)-O2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases). In the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction [Aberg, A., Ormo, M., Nordlund, P., and Sjoberg, B. M. (1993) Biochemistry 32, 9845-9850], implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes. Here we demonstrate that O2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (namely, high concentration of the reductant ascorbate). Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent on the integrity of a hydrogen-bond network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration. These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface.",
author = "Parkin, {Sara E.} and Shuxian Chen and Ley, {Brenda A.} and Lara Mangravite and Edmondson, {Dale E.} and Huynh, {Boi Hanh} and {Bollinger, Jr.}, {Joseph M.}",
year = "1998",
month = "1",
day = "27",
doi = "10.1021/bi9723717",
language = "English (US)",
volume = "37",
pages = "1124--1130",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "4",

}

Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2. / Parkin, Sara E.; Chen, Shuxian; Ley, Brenda A.; Mangravite, Lara; Edmondson, Dale E.; Huynh, Boi Hanh; Bollinger, Jr., Joseph M.

In: Biochemistry, Vol. 37, No. 4, 27.01.1998, p. 1124-1130.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2

AU - Parkin, Sara E.

AU - Chen, Shuxian

AU - Ley, Brenda A.

AU - Mangravite, Lara

AU - Edmondson, Dale E.

AU - Huynh, Boi Hanh

AU - Bollinger, Jr., Joseph M.

PY - 1998/1/27

Y1 - 1998/1/27

N2 - Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122. A key step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O2. As this step leaves only one of the two oxidizing equivalents of the initial diiron(II)-O2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases). In the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction [Aberg, A., Ormo, M., Nordlund, P., and Sjoberg, B. M. (1993) Biochemistry 32, 9845-9850], implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes. Here we demonstrate that O2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (namely, high concentration of the reductant ascorbate). Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent on the integrity of a hydrogen-bond network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration. These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface.

AB - Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122. A key step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O2. As this step leaves only one of the two oxidizing equivalents of the initial diiron(II)-O2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases). In the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction [Aberg, A., Ormo, M., Nordlund, P., and Sjoberg, B. M. (1993) Biochemistry 32, 9845-9850], implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes. Here we demonstrate that O2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (namely, high concentration of the reductant ascorbate). Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent on the integrity of a hydrogen-bond network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration. These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface.

UR - http://www.scopus.com/inward/record.url?scp=0032570317&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032570317&partnerID=8YFLogxK

U2 - 10.1021/bi9723717

DO - 10.1021/bi9723717

M3 - Article

C2 - 9454605

AN - SCOPUS:0032570317

VL - 37

SP - 1124

EP - 1130

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 4

ER -