Structure of the nucleotide radical formed during reaction of CDP/TTP with the E441Q-alpha2beta2 of E. coli ribonucleotide reductase.
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.
Zipse
Hendrik
H
Department of Chemistry and Biochemistry, Ludwig-Maximilians Universitaet Muenchen, 81377 Muenchen, Germany.
Artin
Erin
E
Wnuk
Stanislaw
S
Lohman
Gregory J S
GJ
Martino
Debora
D
Griffin
Robert G
RG
Kacprzak
Sylwia
S
Kaupp
Martin
M
Hoffman
Brian
B
Bennati
Marina
M
Stubbe
Joanne
J
Lees
Nicholas
N
D003565:Cytidine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D003568:Cytidine Monophosphate / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000378:metabolism; D011789:Quantum Theory; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism; D013942:Thymine Nucleotides / Q000737:chemistry* / Q000378:metabolism
D005609:Free Radicals; D013942:Thymine Nucleotides; D003565:Cytidine Diphosphate; D012264:Ribonucleotide Reductases; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase; D003568:Cytidine Monophosphate; C024157:thymidine 5'-triphosphate