Electron paramagnetic resonance (EPR) spectroscopy of the stable-free radical in the native metallo-cofactor of the manganese-ribonucleotide reductase (Mn-RNR) ofCorynebacterium glutamicum

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.

An Active Dimanganese(III)−Tyrosyl Radical Cofactor in Escherichia coli Class Ib Ribonucleotide Reductase

“…In neither case was the yield of active enzyme sufficiently high for biophysical characterization. We propose that we have formed in vitro the same NrdF cofactor isolated from C. ammoniagenes , and perhaps more recently from Corynebacterium glutamicum (14). …”

The alternative aerobic ribonucleotide reductase of Escherichia coli, NrdEF, is a manganese‐dependent enzyme that enables cell replication during periods of iron starvation

“…The induction of mntH is especially pronounced during H 2 O 2 stress, since OxyR directly activates its transcription (Kehres et al ., 2002; Anjem et al ., 2009). Furthermore, in vitro studies have suggested that Corynebacterium glutamicum and Corynebacterium ammoniagenes use manganese for ribonucleotide reductase function (Fieschi et al ., 1998; Abbouni et al ., 2009). If E. coli NrdEF were manganese‐dependent, then the failure of this enzyme to function when the nrdHIEF operon was artificially expressed would be understandable, since manganese is not imported into E. coli under normal growth conditions (Anjem et al ., 2009).…”

Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes

“…Zinc can be erroneously incorporated into the copper-binding protein azurin 39 . Ribonucleotide reductases of various bacteria, upon overexpression in E. coli, contain a dinuclear Fe center, yet in its native state the enzyme possesses a di-manganese center 40,41 . Hence, these data imply that a number of proteins isolated as Zn-binding proteins, may exist as Fe-S proteins inside the cell, emphasizing the importance of assessing the metal occupancy of a protein in its native organism.…”