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.

journal_name

Free Radic Res

journal_title

Free radical research

authors

Griepenburg U,Lassmann G,Auling G

doi

10.3109/10715769609088046

subject

Has Abstract

pub_date

1996-06-01 00:00:00

pages

473-81

issue

6

eissn

1071-5762

issn

1029-2470

journal_volume

24

pub_type

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