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.
Highlights
Ribonucleotide reductase catalyzes the conversion of ribonucleotides to the corresponding deoxyribonucleotides in all living organisms via a radical-based chemical mechanism, thereby providing and controlling the pool of precursors necessary for DNA synthesis and repair [1,2,3,4]
The g1-value from electron paramagnetic resonance (EPR) and HF-EPR, and the rRaman shift indicate that the tyrosyl radical is hydrogen bonded, yet deuterium exchange of the protein had no effect on the rRaman spectra
If a hydrogen bond to the tyrosyl radical is present in Epstein-Barr virus (EBV) R2, our spectroscopic data and DFT analysis indicate that it has characteristics more similar to that in HSV 1 R2, with a hydrogen outside of the phenoxyl plane
Summary
Ribonucleotide reductase catalyzes the conversion of ribonucleotides to the corresponding deoxyribonucleotides in all living organisms via a radical-based chemical mechanism, thereby providing and controlling the pool of precursors necessary for DNA synthesis and repair [1,2,3,4]. Herpes viruses are ubiquitous eukaryotic pathogens infecting a large variety of animal species They share similar architecture with a double-stranded DNA genome encased within a proteinaceous cage. The virus may induce development of several diseases such as infectious mononucleosis, and is associated with neoplasms, including lymphomas and carcinomas [13] In this context, the viral RNR is an important drug target for EBV [14,15]. HF-EPR measurements showed that there are clear differences in the g-tensor anisotropy between different RNR R2s from class Ia [22,23,24] Such differences are assumed to arise from variations in the hydrogen bonding interaction with a nearby hydrogen for both the mouse R2 radical and the YD radical from photo system II (PS II) [22]. We compare our results with previous observations for RNR R2s from mouse, bacteria, and other herpes viruses and discuss possible implications
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