HF-EPR, Raman, UV/VIS Light Spectroscopic, and DFT Studies of the Ribonucleotide Reductase R2 Tyrosyl Radical from Epstein-Barr Virus

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

Human Nail Plate Modifications Induced by Onychomycosis : Implications for Topical Therapy

Open Access - This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are creditedConclusions: Onchomycotic nails presented a thicker but more porous barrier, and its eroded intracellular matrix rendered the tissue more permeable to topically applied chemicals when an aqueous vehicle was used.Purpose: Through the characterisation of the human onchomycotic nail plate this study aimed to inform the design of new topical ungual formulations.Methods: The mechanical properties of the human nail were characterised using a Lloyd tensile strength tester. The nail’s density was determined via pycnometry and the nail’s ultrastructure by electron microscopy. Raman spectroscopy analysed the keratin disulphide bonds within the nail and its permeability properties were assessed by quantifying water and rhodamine uptake.Results: Chronic in vivo nail plate infection increased human nailplate thickness (healthy 0.49 ± 0.15 mm; diseased 1.20 ± 0.67 mm), but reduced its tensile strength (healthy 63.7 ± 13.4 MPa; diseased 41.7 ± 5.0 MPa) and density (healthy 1.34 ± 0.01 g/cm3; diseased 1.29 ± 0.00 g/cm3). Onchomycosis caused cell-cell separation, without disrupting the nail disulfide bonds or desmosomes. The diseased and healthy nails showed equivalent water uptake profiles, but the rhodamine penetration was 4-fold higher in the diseased nails using a PBS vehicle and 3 -fold higher in an ethanol/PBS vehicle.Peer reviewe

A new tyrosyl radical on Phe(208) as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H - Combined X-ray diffraction and EPR/ENDOR studies

The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, (FeFeR)-Fe-III-R-III . Here we report a detailed characterization of center H, using H-1/H-2-N-14/N-15- and Fe-57-ENDOR in comparison with the (FeFeIV)-Fe-III intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe(208) is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe(208) is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical