Electron Paramagnetic Resonance (EPR) Spectroscopy of the Stable-Free Radical in the Native Metallo-Cofactor of the Manganese-Ribonucleotide Reductase (Mn-RNR) of Corynebacterium Glutamicum

Free Radic Res. 2009 Oct;43(10):943-50. doi: 10.1080/10715760903140568. Epub 2009 Aug 21.

Abstract

Ribonucleotide reductases (RNR; EC 1.17.4.1) provide the 2'-deoxyribonucleotides for DNA replication of proliferating cells by a uniform radical mechanism using diverse metals. The native metallo-cofactor of the Corynebacterium glutamicum RNR contains manganese and is sensitive to EDTA and radical scavengers. Hybrid holoenzymes, capable of ribonucleotide reduction, were composed of the small manganese-containing (R2F) and the large catalytic subunit (R1E) from either of the two corynebacterial RNRs. A synthetic peptide deduced from the C-terminal region of the nrdF gene inhibited the C. glutamicum-RNR non-competitively and cross-reacted with the C. ammoniagenes-RNR. The C. glutamicum-R2F has a saturable organic radical signal at g=2.005 detected by electron paramagnetic resonance (EPR) spectroscopy and shows a distinct absorption at 408 nm indicative of a tyrosyl-like organic radical (Y.). Quantification of the metal content revealed 0.06 mol Fe but 0.8 mol Mn per mol R2F-monomer and would thus assign two manganese atoms bound to the dimeric metallo-cofactor, while a distinct enzymatic activity (32 micromol x mg(-1) x min(-1)) was observed in the biochemical complementation assay. Divergence of the C. glutamicum-RNR studied here from the prototypical Salmonella typhimurium class 1b enzyme and the Chlamydia trachomatis class Ic enzyme is discussed below.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Chlamydia trachomatis / enzymology
  • Corynebacterium glutamicum / enzymology*
  • Electron Spin Resonance Spectroscopy
  • Free Radicals / chemistry
  • Manganese / chemistry*
  • Manganese / metabolism
  • Ribonucleotide Reductases / chemistry*
  • Ribonucleotide Reductases / metabolism
  • Salmonella typhimurium / enzymology

Substances

  • Free Radicals
  • Manganese
  • Ribonucleotide Reductases