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Structure of the nucleotide radical formed during reaction of CDP/TTP with the E441Q-alpha2beta2 of E. coli ribonucleotide reductase.

Zipse H, Artin E, Wnuk S, Lohman GJ, Martino D, Griffin RG, Kacprzak S, Kaupp M, Hoffman B, Bennati M, Stubbe J, Lees N - J. Am. Chem. Soc. (2009)

Bottom Line: Incubation of the E441Q-alpha mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale.The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5'-diphosphate.The relationship of this radical to the disulfide radical anion is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Ludwig-Maximilians Universitaet Muenchen, 81377 Muenchen, Germany.

ABSTRACT
The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: alpha and beta. Incubation of the E441Q-alpha mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4'-radical of a 3'-keto-2'-deoxycytidine 5'-diphosphate. To identify the structure of the nucleotide radical [1'-(2)H], [2'-(2)H], [4'-(2)H], [5'-(2)H], [U-(13)C, (15)N], [U-(15)N], and [5,6 -(2)H] CDP and [beta-(2)H] cysteine-alpha were synthesized and incubated with E441Q-alpha2beta2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of (2)H at C4' and C1' altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5'-diphosphate. The relationship of this radical to the disulfide radical anion is discussed.

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Structures considered for II in the quantum chemical calculations.
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fig6: Structures considered for II in the quantum chemical calculations.

Mentions: The assignment was based on the g values obtained from high field EPR (g1 = 2.0072, g2 = 2.0061, and g3 = 2.0021), and the observation of hyperfine interactions with two nuclei (I = 1/2) originally reported by Perrson et al.12,13 In the present paper we describe our efforts using isotopically labeled CDP analogs, E441Q α2, β2, high field EPR, ENDOR analysis and DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) to establish the structure of radical II. The structure is not 5 (Scheme 2) but is proposed to be an off pathway radical with an additional oxygen in the nucleotide (Structure q, Figure 6).


Structure of the nucleotide radical formed during reaction of CDP/TTP with the E441Q-alpha2beta2 of E. coli ribonucleotide reductase.

Zipse H, Artin E, Wnuk S, Lohman GJ, Martino D, Griffin RG, Kacprzak S, Kaupp M, Hoffman B, Bennati M, Stubbe J, Lees N - J. Am. Chem. Soc. (2009)

Structures considered for II in the quantum chemical calculations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651750&req=5

fig6: Structures considered for II in the quantum chemical calculations.
Mentions: The assignment was based on the g values obtained from high field EPR (g1 = 2.0072, g2 = 2.0061, and g3 = 2.0021), and the observation of hyperfine interactions with two nuclei (I = 1/2) originally reported by Perrson et al.12,13 In the present paper we describe our efforts using isotopically labeled CDP analogs, E441Q α2, β2, high field EPR, ENDOR analysis and DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) to establish the structure of radical II. The structure is not 5 (Scheme 2) but is proposed to be an off pathway radical with an additional oxygen in the nucleotide (Structure q, Figure 6).

Bottom Line: Incubation of the E441Q-alpha mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale.The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5'-diphosphate.The relationship of this radical to the disulfide radical anion is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Ludwig-Maximilians Universitaet Muenchen, 81377 Muenchen, Germany.

ABSTRACT
The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: alpha and beta. Incubation of the E441Q-alpha mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4'-radical of a 3'-keto-2'-deoxycytidine 5'-diphosphate. To identify the structure of the nucleotide radical [1'-(2)H], [2'-(2)H], [4'-(2)H], [5'-(2)H], [U-(13)C, (15)N], [U-(15)N], and [5,6 -(2)H] CDP and [beta-(2)H] cysteine-alpha were synthesized and incubated with E441Q-alpha2beta2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of (2)H at C4' and C1' altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5'-diphosphate. The relationship of this radical to the disulfide radical anion is discussed.

Show MeSH
Related in: MedlinePlus