title: Structure of the nitrogen-centered radical formed during inactivation of E. coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine-5'-diphosphate: trapping of the 3'-ketonucleotide.
abstract: Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 has the active site where nucleotide reduction occurs, and R2 contains the diiron tyrosyl radical (Y*) cofactor essential for radical initiation on R1. Mechanism-based inhibitors, such as 2'-azido-2'-deoxyuridine-5'-diphosphate (N(3)UDP), have provided much insight into the reduction mechanism. N(3)UDP is a stoichiometric inactivator that, upon interaction with RNR, results in loss of the Y* in R2 and formation of a nitrogen-centered radical (N*) covalently attached to C225 (R-S-N*-X) in the active site of R1. N(2) is lost prior to N* formation, and after its formation, stoichiometric amounts of 2-methylene-3-furanone, pyrophosphate, and uracil are also generated. On the basis of the hyperfine interactions associated with N*, it was proposed that N* is also covalently attached to the nucleotide through either the oxygen of the 3'-OH (R-S-N*-O-R') or the 3'-C (R-S-N*-C-OH). To distinguish between the proposed structures, the inactivation was carried out with 3'-[(17)O]-N(3)UDP and N* was examined by 9 and 140 GHz EPR spectroscopy. Broadening of the N* signal was detected and the spectrum simulated to obtain the [(17)O] hyperfine tensor. DFT calculations were employed to determine which structures are in best agreement with the simulated hyperfine tensor and our previous ESEEM data. The results are most consistent with the R-S-N*-C-OH structure and provide evidence for the trapping of a 3'-ketonucleotide in the reduction process.
authors.affiliation: Institute of Physical and Theoretical Chemistry, J. W. Goethe University of Frankfurt, and Center for Biological Magnetic Resonance, 60439 Frankfurt, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Fritscher
authors.forename: Erin
authors.initials: E
authors.lastname: Artin
authors.forename: Stanislaw
authors.initials: S
authors.lastname: Wnuk
authors.forename: Galit
authors.initials: G
authors.lastname: Bar
authors.forename: John H
authors.initials: JH
authors.lastname: Robblee
authors.forename: Sylwia
authors.initials: S
authors.lastname: Kacprzak
authors.forename: Martin
authors.initials: M
authors.lastname: Kaupp
authors.forename: Robert G
authors.initials: RG
authors.lastname: Griffin
authors.forename: Marina
authors.initials: M
authors.lastname: Bennati
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D001386:Azides / Q000737:chemistry* / Q000494:pharmacology*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry* / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D011789:Quantum Theory; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001386:Azides; D003856:Deoxyuracil Nucleotides; D009711:Nucleotides; C053847:2'-azido-2'-deoxyuridine 5'-diphosphate; D012264:Ribonucleotide Reductases