title: Detection of a stable free radical in the B2 subunit of the manganese ribonucleotide reductase (Mn-RRase) of Corynebacterium ammoniagenes.
abstract: Ribonucleotide reductases catalyze the irreversible reductive formation of 2'-deoxyribonucleotides required for DNA replication and cell proliferation, and a radical mechanism was assumed to be involved in this reaction. In order to search for a radical in the aerobic manganese ribonucleotide reductase (Mn-RRase) by electron paramagnetic resonance (EPR) the native metal-containing 100 kDa B2 subunit was deliberately prepared from the wild type strain Corynebacterium ammoniagenes ATCC 6872. Enrichment by 2'5'-ADP Sepharose 4B affinity chromatography, fast protein liquid chromatography (FPLC) with SuperoseTM12 and concentration by vacuum evaporation allowed for the first time the detection of a stable free radical by EPR spectroscopy at 77 K. The EPR spectrum exhibits an easily saturable doublet of 1.8 mT splitting and a line width of 1.3 mT at g = 2.0040. The EPR signal intensity showed a clear correlation with the enzymatic activity upon long-time storage at ambient temperature (294 K) and inactivation by the specific RRase inhibitor hydroxyurea (HU). This leads to the assumption of a protein-linked radical, with functional significance, in the metal-containing 100 kDa B2 subunit of the MnRRase of Corynebacterium ammoniagenes.
authors.affiliation: Fachbereich Biologie-Chemie der Universität, Kassel, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Griepenburg
authors.forename: G
authors.initials: G
authors.lastname: Lassmann
authors.forename: G
authors.initials: G
authors.lastname: Auling
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D003352:Corynebacterium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D008345:Manganese; D012264:Ribonucleotide Reductases / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D005609:Free Radicals; D008345:Manganese; D012264:Ribonucleotide Reductases