title: HF-EPR, Raman, UV/VIS light spectroscopic, and DFT studies of the ribonucleotide reductase R2 tyrosyl radical from Epstein-Barr virus.
abstract: Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g₁-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm⁻¹) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe²⁺ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class.
authors: Department of Molecular Biosciences, University of Oslo, Oslo, Norway.
authors: Ane B
authors: AB
authors: Tomter
authors: Giorgio
authors: G
authors: Zoppellaro
authors: Florian
authors: F
authors: Schmitzberger
authors: Niels H
authors: NH
authors: Andersen
authors: Anne-Laure
authors: AL
authors: Barra
authors: Henrik
authors: H
authors: Engman
authors: Pär
authors: P
authors: Nordlund
authors: K Kristoffer
authors: KK
authors: Andersson
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005609:Free Radicals / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D012264:Ribonucleotide Reductases; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman / Q000379:methods*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D014443:Tyrosine; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit