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Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.

Tomter AB, Zoppellaro G, Bell CB, Barra AL, Andersen NH, Solomon EI, Andersson KK - PLoS ONE (2012)

Bottom Line: Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides.The two similar X-band EPR spectra did not change significantly over 4 to 50 K.This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g(1)-value of 2.0090 for the tyrosyl radical was extracted. This g(1)-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange. Additionally, the (18)O-sensitive Fe-O-Fe symmetric stretching (483 cm(-1)) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g(1)-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053-33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.

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The X-band (9.66 GHz) EPR spectrum of R2F-MnIII2-Tyr•B. cereus R2F (200 µM R2F) reconstituted in presence of 2X NrdIhq.Recorded at T = 20 K, 16 µW microwave power, 0.2 mT modulation amplitude, and 4 scans (Obs) and its spectrum simulation (Sim).
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pone-0033436-g006: The X-band (9.66 GHz) EPR spectrum of R2F-MnIII2-Tyr•B. cereus R2F (200 µM R2F) reconstituted in presence of 2X NrdIhq.Recorded at T = 20 K, 16 µW microwave power, 0.2 mT modulation amplitude, and 4 scans (Obs) and its spectrum simulation (Sim).

Mentions: The X-band EPR spectrum of the tyrosyl radical in R2F-MnIII2-Tyr•B. cereus R2F recorded at (T = 20 K) is shown in Figure 6 (upper line, Obs) together with one simulation (lower line, Sim). The recorded resonance and derived g-tensor parameters (using spectrum simulation, g1 = 2.0094, g2 = 2.0039, g3 = 2.0022), thus are only tentative but show alteration of the magnetic fingerprints of the tyrosyl radical when manganese-ions replaces two iron ions. Furthermore, the loss of resolved AH-hyperfine resonance signals accompanied by signal broadening (∼8.5 mT) is evident in the R2F-MnIII2-Tyr• spectrum. The list of R2F-MnIII2-Tyr• EPR simulation parameters (values enclosed in parentheses, tentative) are given in Table 1. The dihedral angle (θ) of the tyrosyl radical in B. cereus R2F-MnIII2-Tyr• was estimated as ∼65°. The spin concentration of the tyrosyl radical was determined in this case as ∼0.25 spin per dimer. The g1 resonance feature of the B. cereus R2F-FeIII2-Tyr• HF-EPR signal can also be observed in the HF-EPR spectrum of R2F-MnIII2-Tyr• (Figure S2, T = 5 K). However, the entire g-tensor values of R2F-MnIII2-Tyr•, estimated from X-band measurements, is poorly resolved in the HF-EPR spectrum. This is due to low radical concentration and the presence of manganese impurities overlapping on g2 and g3. HF-EPR is very sensitive to Mn(II) impurities and removal of most of the Mn(II) by gelfiltation was needed, which lower the tyrosyl-radical content in R2F-MnIII2-Tyr• and at 5 K the two Mn(II) impurities (Figure S2) are partially saturated. At 10 K or 15 K the R2F-MnIII2-Tyr• radical 285 GHz spectrum, is much more dominated by the Mn(II) impurities. Our findings indicate that presence of Mn(II) impurities can only be partly removed from the sample, but at the cost of a substantial loss in radical yield and resolution of the g-tensor.


Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.

Tomter AB, Zoppellaro G, Bell CB, Barra AL, Andersen NH, Solomon EI, Andersson KK - PLoS ONE (2012)

The X-band (9.66 GHz) EPR spectrum of R2F-MnIII2-Tyr•B. cereus R2F (200 µM R2F) reconstituted in presence of 2X NrdIhq.Recorded at T = 20 K, 16 µW microwave power, 0.2 mT modulation amplitude, and 4 scans (Obs) and its spectrum simulation (Sim).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303829&req=5

pone-0033436-g006: The X-band (9.66 GHz) EPR spectrum of R2F-MnIII2-Tyr•B. cereus R2F (200 µM R2F) reconstituted in presence of 2X NrdIhq.Recorded at T = 20 K, 16 µW microwave power, 0.2 mT modulation amplitude, and 4 scans (Obs) and its spectrum simulation (Sim).
Mentions: The X-band EPR spectrum of the tyrosyl radical in R2F-MnIII2-Tyr•B. cereus R2F recorded at (T = 20 K) is shown in Figure 6 (upper line, Obs) together with one simulation (lower line, Sim). The recorded resonance and derived g-tensor parameters (using spectrum simulation, g1 = 2.0094, g2 = 2.0039, g3 = 2.0022), thus are only tentative but show alteration of the magnetic fingerprints of the tyrosyl radical when manganese-ions replaces two iron ions. Furthermore, the loss of resolved AH-hyperfine resonance signals accompanied by signal broadening (∼8.5 mT) is evident in the R2F-MnIII2-Tyr• spectrum. The list of R2F-MnIII2-Tyr• EPR simulation parameters (values enclosed in parentheses, tentative) are given in Table 1. The dihedral angle (θ) of the tyrosyl radical in B. cereus R2F-MnIII2-Tyr• was estimated as ∼65°. The spin concentration of the tyrosyl radical was determined in this case as ∼0.25 spin per dimer. The g1 resonance feature of the B. cereus R2F-FeIII2-Tyr• HF-EPR signal can also be observed in the HF-EPR spectrum of R2F-MnIII2-Tyr• (Figure S2, T = 5 K). However, the entire g-tensor values of R2F-MnIII2-Tyr•, estimated from X-band measurements, is poorly resolved in the HF-EPR spectrum. This is due to low radical concentration and the presence of manganese impurities overlapping on g2 and g3. HF-EPR is very sensitive to Mn(II) impurities and removal of most of the Mn(II) by gelfiltation was needed, which lower the tyrosyl-radical content in R2F-MnIII2-Tyr• and at 5 K the two Mn(II) impurities (Figure S2) are partially saturated. At 10 K or 15 K the R2F-MnIII2-Tyr• radical 285 GHz spectrum, is much more dominated by the Mn(II) impurities. Our findings indicate that presence of Mn(II) impurities can only be partly removed from the sample, but at the cost of a substantial loss in radical yield and resolution of the g-tensor.

Bottom Line: Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides.The two similar X-band EPR spectra did not change significantly over 4 to 50 K.This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g(1)-value of 2.0090 for the tyrosyl radical was extracted. This g(1)-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange. Additionally, the (18)O-sensitive Fe-O-Fe symmetric stretching (483 cm(-1)) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g(1)-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053-33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.

Show MeSH
Related in: MedlinePlus