Each beta-protomer of the small beta beta subunit of Escherichia coli ribonucleotide reductase (R2) contains a binuclear iron cluster with inequivalent binding sites: Fe-A and Fe-B. In anaerobic Fe-II titrations of apoprotein under standard buffer conditions, we show that the majority of the protein binds only one Fe-II atom per beta beta subunit. Additional iron occupation can be achieved upon exposure to O-2 or in high glycerol buffers. The differential binding affinity of the A- and B-sites allows us to produce heterobinuclear (MnFeII)-Fe-II and novel (MnFeIII)-Fe-III clusters within a single beta-protomer of R2. The oxidized species are produced with H2O2 addition. We demonstrate that no significant exchange of metal occurs between the A-and B-sites, and thus the binding of the first metal is under kinetic control, as has been suggested previously. The binding of first Fell atom to the active site in a beta-protomer (beta(I)) induces a global protein conformational change that inhibits access of metal to the active site in the other beta-protomer (beta(II)). The binding of the same Fe-II atom also induces a local effect at the active site in beta(1)-protomer, which lowers the affinity for metal in the A-site. The mixed metal FeMn species are quantitatively characterized with electron paramagnetic resonance spectroscopy. The previously reported catalase activity of Mn(2)(II)R2 is shown not to be associated with Mn.