The active form of the Saccharomyces cerevisiae ribonucleotide reductase small subunit is a heterodimer in vitro and in vivo.

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: RI and R2. R2 houses a diferric-tyrosyl radical (Y-.) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (beta(2)) and Y4 (beta'2). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (beta beta') is thought to be the active form, several rnr4 Delta 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-(HiS(6))-beta(2) ((His)beta(2)) is mixed with beta'2, apo-(His)beta beta' forms quantitatively within 2 min. In contrast, holo-beta beta' fails to exchange with apo-(His)beta(2) to form holo-(His)beta beta 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 beta beta' is the active form. The catalytic activity, protein concentrations, and Y-. content of the rnr4 Delta and wild type (wt) strains were compared to clarify the role of beta' in vivo. The Y-. content of rnr4 Delta is 15-fold less than that of wt, consistent with the observed low activity of rnr4 Delta extracts (<0.01 nmol min(-1) mg(-1)) versus wt (0.06 +/- 0.01 nmol min(-1) mg(-1)). FLAG 02 isolated from the rnr4 Delta strain has a specific activity of 2 nmol min(-1) mg(-1), similar to that of reconstituted apo(His)beta(2) (10 nmol min-(1) mg(-1)), but significantly less than holo-(His)beta beta' (similar to 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 beta beta'.