Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of E. coli Ribonucleotide Reductase
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- 作者：Hendrik Zipse ; Erin Artin ; Stanislaw Wnuk ; Gregory J. S. Lohman ; Debora Martino ; Robert G. Griffin ; Sylwia Kacprzak ; Martin Kaupp ; Brian Hoffman ; Marina Bennati ; JoAnne Stubbe ; Nicholas Lees
- 刊名：Journal of the American Chemical Society
- 出版年：2009
- 出版时间：January 14, 2009
- 年：2009
- 卷：131
- 期：1
- 页码：200-211
- 全文大小：332K
- 年卷期：v.131,no.1(January 14, 2009)
- ISSN：1520-5126
文摘
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: α and β. Incubation of the E441Q-α 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′-2H], [2′-2H], [4′-2H], [5′-2H], [U−13C, 15N], [U−15N], and [5,6 -2H] CDP and [β-2H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of 2H 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.