The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y*) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (beta2) and Y4 (beta'2). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (betabeta') is thought to be the active form, several rnr4delta strains are viable. To resolve this paradox, N-terminally epitope-tagged beta and beta' were expressed in E. coli or integrated into the yeast genome. In vitro exchange studies reveal that when apo-(His6)-beta2 ((His)beta2) is mixed with beta'2, apo-(His)betabeta' forms quantitatively within 2 min. In contrast, holo-betabeta' fails to exchange with apo-(His)beta2 to form holo-(His)betabeta and beta'2. Isolation of genomically encoded tagged beta or beta' from yeast extracts gave a 1:1 complex of beta and beta', suggesting that betabeta' is the active form. The catalytic activity, protein concentrations, and Y* content of the rnr4delta and wild type (wt) strains were compared to clarify the role of beta' in vivo. The Y* content of rnr4delta is 15-fold less than that of wt, consistent with the observed low activity of rnr4delta extracts (<0.01 nmol min(-1) mg(-1)) versus wt (0.06 +/- 0.01 nmol min(-1) mg(-1)). (FLAG)beta2 isolated from the rnr4delta strain has a specific activity of 2 nmol min(-1) mg(-1), similar to that of reconstituted apo-(His)beta2 (10 nmol min(-1) mg(-1)), but significantly less than holo-(His)betabeta' (approximately 2000 nmol min(-1) mg(-1)). These studies together demonstrate that beta' plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is betabeta'.