For deoxyribonucleotide synthesis during anaerobic growth, Escherichia coli cells depend on an oxygen-sensitive class III ribonucleotide reductase. The enzyme system consists of two proteins: protein α, on which ribonucleotides bind and are reduced, and protein β, of which the function is to introduce a catalytically essential glycyl radical on protein α. Protein β can assemble one [4Fe-4S] center per polypeptide enjoying both the [4Fe-4S]2+ and [4Fe-4S]1+ redox state, as shown by iron and sulfide analysis, Mössbauer spectroscopy (δ = 0.43 mm·s−1, ΔE Q= 1.0 mm·s−1, [4Fe-4S]2+), and EPR spectroscopy (g = 2.03 and 1.93, [4Fe-4S]1+). This iron center is sensitive to oxygen and can decompose into stable [2Fe-2S]2+ centers during exposure to air. This degraded form is nevertheless active, albeit to a lesser extent because of the conversion of the cluster into [4Fe-4S] forms during the strongly reductive conditions of the assay. Furthermore, protein β has the potential to activate several molecules of protein α, suggesting that protein β is an activating enzyme rather than a component of an α2β2 complex as previously claimed.