Each beta-protomer of the small betabeta 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 betabeta subunit. Additional iron occupation can be achieved upon exposure to O2 or in high glycerol buffers. The differential binding affinity of the A- and B-sites allows us to produce heterobinuclear Mn(II)Fe(II) and novel Mn(III)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 Fe(II) atom to the active site in a beta-protomer (betaI) induces a global protein conformational change that inhibits access of metal to the active site in the other beta-protomer (betaII). The binding of the same Fe(II) atom also induces a local effect at the active site in betaI-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 Mn2(II)R2 is shown not to be associated with Mn.