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

Ane B Tomter,Edward I Solomon,Niels H Andersen,Anne-Laure Barra,K Kristoffer Andersson,Giorgio Zoppellaro,Caleb B Bell

doi:10.1371/journal.pone.0033436

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, ν7a = 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 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.

Highlights

Ribonucleotide reductases (RNRs) catalyze the reduction of the four ribonucleotides to the corresponding deoxyribonucleotides, providing the precursors for the DNA synthesis and repair in all living organisms [1,2,3,4,5]
The radical signal completely vanished after 5 min when the active Fe loaded protein was treated with 4 mM hydroxyurea (HU), which is a common radical scavenger for RNR R2 (Figure 2A, red line)
The Electron Paramagnetic Resonance (EPR) resonance fingerprint and temperature behavior of R2FMnIII2-TyrN are different from those observed in the Mn forms of E. coli, C. ammoniagenes and the recently discovered B. subtilis [22].This argues for a weaker magnetic interaction between radical and metal sites in B. cereus, relative to those in E. coli and C. ammoniagenes RNR R2 proteins

Summary

Introduction

Ribonucleotide reductases (RNRs) catalyze the reduction of the four ribonucleotides to the corresponding deoxyribonucleotides, providing the precursors for the DNA synthesis and repair in all living organisms [1,2,3,4,5]. This step is an attractive target for drug design strategies against rapidly proliferating cells such as cancers and various pathogens, as it is the rate limiting step in the DNA synthesis [6]. The generated R2 radical is shuttled approximately 35 Ato the active site of the R1 subunit where it forms a thiyl radical, through a proposed conserved network of hydrogen bonded amino acids [4,7,12,13,14]

Methods

Results

Conclusion