Spectroscopic Studies of the Iron- and Manganese Reconstituted Tyrosyl Radical in Bacillus cereus Ribonucleotide Reductase R2 Protein

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 ribonucleotide reduction active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. WIn this work, we studied the R2F subunit of B. cereus reconstituted with both iron and or alternatively with manganese ions, then subsequently reacted with molecular . oxygen to generate tyrosyl-radicals. The catalytically active tyrosyl radical forms were probed by EPR, and resonance Raman spectroscopy. The X-band spectrum of the RNR R2F tyrosyl radical reconstituted with iron was slightly different from that reconstituted with manganese.T However, the two similar X-band EPR spectra of both did not change significantly over the temperature range examined (4-50 K). From the 285 GHz EPR spectrum of the iron form, a g1-value of 2.0090 for the tyrosyl radical was extracted. This g1-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, 7a = 1500 cm-1) was found to be insensitive to deuterium-oxide exchange. Additionally, the 18O-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 bond cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g1-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 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) results indicate that both the manganese and iron reconstituted RNR R2F could beare functional and the manganese form might be very importantmost significant, as it has 8 times higher activity.