Mössbauer and EPR studies of the binuclear iron center in ribonucleotide reductase from Escherichia coli

57Fe-enriched ribonucleotide reductase subunit B2 from Escherichia colistrain N6405/pSPS2 has been characterized by Mössbauer and EPR spectroscopy in its native diferric state and in a new differous form. The native protein exhibits two Mössbauer doublets in a 1:1 ratio with parameters that are in excellent agreement with those reported for the wild-type protein (Atkin, C. L., Thelander, L., Reichard, P., and Lang, G. (1983) J. Biol. Chem.248, 7464–7472); in addition, our studies show the absence of adventitiously bound iron. The iron content in the present samples approached 4 per B2 subunit, and the tyrosyl radical content exceeded 1 per B2 subunit. The higher values are attributed to the use of a new ϵ280for the protein and more efficient methods for iron extraction. We thus propose that subunit B2 has two binuclear iron clusters, each associated with its own tyrosyl radical, in contradistinction from the prevailing model.