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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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Mentions: The 13C hyperfine tensors thus arrived at are quite different from earlier experimental studies of reference systems such as malonic acid (A = [30, 30, 210] MHz,(53) or CH2OH radical (aiso ≈ 130 MHz,(54) Figure 8). Our observation is consistent with a recent report of a semidione radical,(55) in which the 13C hyperfine couplings also appeared to be different from those expected for carbon-centered radicals. This led us to test the validity of the computed tensors for malonic acid and CH2OH radicals, and of the unsubstituted semidione radical by use of the ADF DFT package as well as Gaussian 03 to calculate the 13C couplings (Figure 8, Table 3). The hyperfine couplings obtained from both packages for the malonic acid and CH2OH radicals agreed satisfactorily with experiment and with each other, while the ADF value for the semidione radical are quite similar to those calculated for II with Gaussian 03.


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)

© Copyright Policy - open-access
Related In: Results  -  Collection

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

Mentions: The 13C hyperfine tensors thus arrived at are quite different from earlier experimental studies of reference systems such as malonic acid (A = [30, 30, 210] MHz,(53) or CH2OH radical (aiso ≈ 130 MHz,(54) Figure 8). Our observation is consistent with a recent report of a semidione radical,(55) in which the 13C hyperfine couplings also appeared to be different from those expected for carbon-centered radicals. This led us to test the validity of the computed tensors for malonic acid and CH2OH radicals, and of the unsubstituted semidione radical by use of the ADF DFT package as well as Gaussian 03 to calculate the 13C couplings (Figure 8, Table 3). The hyperfine couplings obtained from both packages for the malonic acid and CH2OH radicals agreed satisfactorily with experiment and with each other, while the ADF value for the semidione radical are quite similar to those calculated for II with Gaussian 03.

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