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

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

Vaccination against hepatitis B with 4-double doses increases response rates and antibodies titers in HIV-infected adults

Submitted by Sandra Infurna ([email protected]) on 2017-01-17T14:11:55Z No. of bitstreams: 1 jc_miguel_etal_IOC_2012.pdf: 346787 bytes, checksum: 7a75068f25ae94e6e7e0bb55614fc4f4 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-01-17T14:21:49Z (GMT) No. of bitstreams: 1 jc_miguel_etal_IOC_2012.pdf: 346787 bytes, checksum: 7a75068f25ae94e6e7e0bb55614fc4f4 (MD5)Made available in DSpace on 2017-01-17T14:21:49Z (GMT). No. of bitstreams: 1 jc_miguel_etal_IOC_2012.pdf: 346787 bytes, checksum: 7a75068f25ae94e6e7e0bb55614fc4f4 (MD5) Previous issue date: 2012Universidade Federal do Rio de Janeiro. Faculdade de Medicina. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Escola Nacional de Saúde Pública Sérgio Arouca. Rio de Janeiro, RJ, Brasil.Universidade Federal Fluminense. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Referência Nacional Para Hepatites Virais. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Referência Nacional Para Hepatites Virais. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Referência Nacional Para Hepatites Virais. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Referência Nacional Para Hepatites Virais. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Faculdade de Medicina. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Faculdade de Medicina. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Faculdade de Medicina. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil.Background: Antibody responses to standard regimens of hepatitis B (HBV) vaccination are lower in HIVinfected subjects and the best hepatitis B vaccine schedule in this population is not known. Objective: To assess the immunogenicity and to evaluate predictors of serologic response of a modified regimen of a HBV recombinant vaccine in a cohort of HIV-infected subjects. Methods: HIV-infected subjects received 4 doses (40 g) of a recombinant HBV vaccine at 0, 1, 2 and 6 months. Demographic information as well as CD4 cell count and plasma viral load were assessed at baseline. Protective and strong responses were defined as an anti-HBs titer ≥10 mIU/mL and ≥100 mIU/mL, respectively and were evaluated one month after the third and the fourth doses. Results: 163 HIV-infected individuals were evaluated 67 (40%) were male and median age was 37 years. Median CD4 cell count was 385 cells/mm3 and 113 (70%) had undetectable HIV-1 viral load. Protective antibody response was observed in 83 and 91% and a strong antibody response was observed in 62 and 80% of the subjects after 3 and 4 doses, respectively. In a multivariate logistic model undetectable HIV-1 viral load and higher CD4 cell counts were independent predictors of a strong antibody response after 4 doses. Patients with undetectable HIV viral load were almost 3 times more likely to have anti-HBs titers above 100 mIU/mL than those with detectable viral load. Conclusions: A 4-double-dose regimen of a recombinant HBV vaccine increased response rates and determined higher antibody titers which may translate in prolonged protection agains HBV. Inclusion of a fourth dose of HBV vaccine for HIV-infected subjects should be considered in the public health setting