EPR investigations of the inactivation of E. coli ribonucleotide reductase with 2′-azido-2′-deoxyuridine 5′-diphosphate: Evidence for the involvement of the thiyl radical of C225-R1

Wilfred A. Van Der Donk, JoAnne Stubbe, Gary J. Gerfen, Brendan F. Bellew, Robert G. Griffin

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. 2′-Azido-2′-deoxyuridine 5′-diphosphate (N3UDP) has previously been shown to be a stoichiometric mechanism based inhibitor of this enzyme. Inactivation of RNR is accompanied by loss of the tyrosyl radical on the R2 subunit concomitant with formation of a new nitrogen centered radical. The X-band EPR spectrum of this radical species exhibits a triplet hyperfine interaction of ∼25 G arising from one of the three nitrogens of the azide moiety of N3UDP and a doublet hyperfine interaction of 6.3 G which has been proposed to arise from a proton. High frequency (139.5 GHz) EPR spectroscopic studies of this nitrogen centered radical have resolved the peaks corresponding to all three principal g-values: g11 = 2.01557, g22 = 2.00625, and g33 = 2.00209. In addition, the nitrogen hyperfine splitting along g33 is resolved (A33N = 31.0 G) and upper limits (∼5 G) can be placed on both A11N and A22N. Comparison of these g- and A-values with those of model systems in the literature suggests a structure for the radical, XNSCH2-, in which SCH2 is part of a cysteine residue of R1, and X is either a nonprotonated sulfur, oxygen, or carbon moiety. Use of an E. coli strain that is auxotrophic for cysteine and contains the nucleotide reductase gene allowed [β-2H]cysteine labeled RNR to be prepared. Incubation of this isotopically labeled protein with N3UDP produced the radical signal without the hyperfine splitting of 6.3 G, indicating that this interaction is associated with a proton from the -SCH2- component of the proposed structure. These results establish that the nitrogen centered radical is covalently attached to a cysteine, probably C225, of the R1 subunit of RNR. Site-directed mutagenesis studies with a variety of R1 mutants in which each cysteine (439, 462, 754, and 759) was converted to a serine reveal that X cannot be a substituted sulfur. A structure for the nitrogen centered radical is proposed in which X is derived from 3′-keto-2′-deoxyuridine 5′-diphosphate, an intermediate in the inactivation of RNR by N3UDP. Specifically, X is proposed to be the 3′-hydroxyl oxygen of the deoxyribose moiety.

Original languageEnglish (US)
Pages (from-to)8908-8916
Number of pages9
JournalJournal of the American Chemical Society
Volume117
Issue number35
StatePublished - Sep 6 1995
Externally publishedYes

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Ribonucleotide Reductases
Escherichia coli
Paramagnetic resonance
Nitrogen
Cysteine
Nucleotides
Sulfur
Protons
Oxygen
Deoxyribose
Deoxyuridine
Mutagenesis
Azides
Diphosphates
Enzyme Inhibitors
Site-Directed Mutagenesis
Hydroxyl Radical
Serine
2'-azido-2'-deoxyuridine 5'-diphosphate
Cetuximab

ASJC Scopus subject areas

  • Chemistry(all)

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EPR investigations of the inactivation of E. coli ribonucleotide reductase with 2′-azido-2′-deoxyuridine 5′-diphosphate : Evidence for the involvement of the thiyl radical of C225-R1. / Van Der Donk, Wilfred A.; Stubbe, JoAnne; Gerfen, Gary J.; Bellew, Brendan F.; Griffin, Robert G.

In: Journal of the American Chemical Society, Vol. 117, No. 35, 06.09.1995, p. 8908-8916.

Research output: Contribution to journalArticle

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title = "EPR investigations of the inactivation of E. coli ribonucleotide reductase with 2′-azido-2′-deoxyuridine 5′-diphosphate: Evidence for the involvement of the thiyl radical of C225-R1",
abstract = "Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. 2′-Azido-2′-deoxyuridine 5′-diphosphate (N3UDP) has previously been shown to be a stoichiometric mechanism based inhibitor of this enzyme. Inactivation of RNR is accompanied by loss of the tyrosyl radical on the R2 subunit concomitant with formation of a new nitrogen centered radical. The X-band EPR spectrum of this radical species exhibits a triplet hyperfine interaction of ∼25 G arising from one of the three nitrogens of the azide moiety of N3UDP and a doublet hyperfine interaction of 6.3 G which has been proposed to arise from a proton. High frequency (139.5 GHz) EPR spectroscopic studies of this nitrogen centered radical have resolved the peaks corresponding to all three principal g-values: g11 = 2.01557, g22 = 2.00625, and g33 = 2.00209. In addition, the nitrogen hyperfine splitting along g33 is resolved (A33N = 31.0 G) and upper limits (∼5 G) can be placed on both A11N and A22N. Comparison of these g- and A-values with those of model systems in the literature suggests a structure for the radical, XN•SCH2-, in which SCH2 is part of a cysteine residue of R1, and X is either a nonprotonated sulfur, oxygen, or carbon moiety. Use of an E. coli strain that is auxotrophic for cysteine and contains the nucleotide reductase gene allowed [β-2H]cysteine labeled RNR to be prepared. Incubation of this isotopically labeled protein with N3UDP produced the radical signal without the hyperfine splitting of 6.3 G, indicating that this interaction is associated with a proton from the -SCH2- component of the proposed structure. These results establish that the nitrogen centered radical is covalently attached to a cysteine, probably C225, of the R1 subunit of RNR. Site-directed mutagenesis studies with a variety of R1 mutants in which each cysteine (439, 462, 754, and 759) was converted to a serine reveal that X cannot be a substituted sulfur. A structure for the nitrogen centered radical is proposed in which X is derived from 3′-keto-2′-deoxyuridine 5′-diphosphate, an intermediate in the inactivation of RNR by N3UDP. Specifically, X is proposed to be the 3′-hydroxyl oxygen of the deoxyribose moiety.",
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AU - Van Der Donk, Wilfred A.

AU - Stubbe, JoAnne

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N2 - Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. 2′-Azido-2′-deoxyuridine 5′-diphosphate (N3UDP) has previously been shown to be a stoichiometric mechanism based inhibitor of this enzyme. Inactivation of RNR is accompanied by loss of the tyrosyl radical on the R2 subunit concomitant with formation of a new nitrogen centered radical. The X-band EPR spectrum of this radical species exhibits a triplet hyperfine interaction of ∼25 G arising from one of the three nitrogens of the azide moiety of N3UDP and a doublet hyperfine interaction of 6.3 G which has been proposed to arise from a proton. High frequency (139.5 GHz) EPR spectroscopic studies of this nitrogen centered radical have resolved the peaks corresponding to all three principal g-values: g11 = 2.01557, g22 = 2.00625, and g33 = 2.00209. In addition, the nitrogen hyperfine splitting along g33 is resolved (A33N = 31.0 G) and upper limits (∼5 G) can be placed on both A11N and A22N. Comparison of these g- and A-values with those of model systems in the literature suggests a structure for the radical, XN•SCH2-, in which SCH2 is part of a cysteine residue of R1, and X is either a nonprotonated sulfur, oxygen, or carbon moiety. Use of an E. coli strain that is auxotrophic for cysteine and contains the nucleotide reductase gene allowed [β-2H]cysteine labeled RNR to be prepared. Incubation of this isotopically labeled protein with N3UDP produced the radical signal without the hyperfine splitting of 6.3 G, indicating that this interaction is associated with a proton from the -SCH2- component of the proposed structure. These results establish that the nitrogen centered radical is covalently attached to a cysteine, probably C225, of the R1 subunit of RNR. Site-directed mutagenesis studies with a variety of R1 mutants in which each cysteine (439, 462, 754, and 759) was converted to a serine reveal that X cannot be a substituted sulfur. A structure for the nitrogen centered radical is proposed in which X is derived from 3′-keto-2′-deoxyuridine 5′-diphosphate, an intermediate in the inactivation of RNR by N3UDP. Specifically, X is proposed to be the 3′-hydroxyl oxygen of the deoxyribose moiety.

AB - Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. 2′-Azido-2′-deoxyuridine 5′-diphosphate (N3UDP) has previously been shown to be a stoichiometric mechanism based inhibitor of this enzyme. Inactivation of RNR is accompanied by loss of the tyrosyl radical on the R2 subunit concomitant with formation of a new nitrogen centered radical. The X-band EPR spectrum of this radical species exhibits a triplet hyperfine interaction of ∼25 G arising from one of the three nitrogens of the azide moiety of N3UDP and a doublet hyperfine interaction of 6.3 G which has been proposed to arise from a proton. High frequency (139.5 GHz) EPR spectroscopic studies of this nitrogen centered radical have resolved the peaks corresponding to all three principal g-values: g11 = 2.01557, g22 = 2.00625, and g33 = 2.00209. In addition, the nitrogen hyperfine splitting along g33 is resolved (A33N = 31.0 G) and upper limits (∼5 G) can be placed on both A11N and A22N. Comparison of these g- and A-values with those of model systems in the literature suggests a structure for the radical, XN•SCH2-, in which SCH2 is part of a cysteine residue of R1, and X is either a nonprotonated sulfur, oxygen, or carbon moiety. Use of an E. coli strain that is auxotrophic for cysteine and contains the nucleotide reductase gene allowed [β-2H]cysteine labeled RNR to be prepared. Incubation of this isotopically labeled protein with N3UDP produced the radical signal without the hyperfine splitting of 6.3 G, indicating that this interaction is associated with a proton from the -SCH2- component of the proposed structure. These results establish that the nitrogen centered radical is covalently attached to a cysteine, probably C225, of the R1 subunit of RNR. Site-directed mutagenesis studies with a variety of R1 mutants in which each cysteine (439, 462, 754, and 759) was converted to a serine reveal that X cannot be a substituted sulfur. A structure for the nitrogen centered radical is proposed in which X is derived from 3′-keto-2′-deoxyuridine 5′-diphosphate, an intermediate in the inactivation of RNR by N3UDP. Specifically, X is proposed to be the 3′-hydroxyl oxygen of the deoxyribose moiety.

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