Ribonucleotide reduction, the unique step in the pathway to DNA synthesis, is catalyzed by enzymes via radical‐dependent redox chemistry involving an array of diverse metallocofactors. The nucleotide reduction gene (nrdF) encoding the metallocofactor containing small subunit (R2F) of the Corynebacterium ammoniagenes ribonucleotide reductase was reintroduced into strain C. ammoniagenes ATCC 6872. Efficient homologous expression from plasmid pOCA2 using the tac‐promotor enabled purification of R2F to homogeneity. The chromatographic protocol provided native R2F with a high ratio of manganese to iron (30 : 1), high activity (69 μmol 2′‐deoxyribonucleotide·mg−1·min−1) and distinct absorption at 408 nm, characteristic of a tyrosyl radical (Y˙), which is sensitive to the radical scavenger hydroxyurea. A novel enzyme assay revealed the direct involvement of Y˙ in ribonucleotide reduction because 0.2 nmol 2′‐deoxyribonucleotide was formed, driven by 0.4 nmol Y˙ located on R2F. X‐band electron paramagnetic resonance spectroscopy demonstrated a tyrosyl radical at an effective g‐value of 2.004. Temperature dependent X/Q‐band EPR studies revealed that this radical is coupled to a metallocofactor. Similarities of the native C. ammoniagenes ribonucleotide reductase to the in vitro activated Escherichia coli class Ib enzyme containing a dimanganese(III)‐tyrosyl metallocofactor are discussed.

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