The Active Form of the Saccharomyces cerevisiae Ribonucleotide

FLAGβ2 isolated from the rnr4Δ strain has a specific activity of 2 nmol min-1 mg-1, similar to that of reconstituted apo-Hisβ2 (10 nmol min-1 mg-1)...
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15366

Biochemistry 2005, 44, 15366-15377

The Active Form of the Saccharomyces cereVisiae Ribonucleotide Reductase Small Subunit Is a Heterodimer in Vitro and in ViVo† Deborah L. Perlstein,‡,§ Jie Ge,|,§ Allison D. Ortigosa,§ John H. Robblee,§ Zhen Zhang,⊥ Mingxia Huang,⊥ and JoAnne Stubbe*,§ Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Biochemistry and Molecular Genetics, UniVersity of Colorado Health Sciences Center, Aurora, Colorado 80045 ReceiVed August 13, 2005; ReVised Manuscript ReceiVed September 15, 2005

ABSTRACT: 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 (β2) and Y4 (β′2). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (ββ′) is thought to be the active form, several rnr4∆ strains are viable. To resolve this paradox, N-terminally epitope-tagged β and β′ were expressed in E. coli or integrated into the yeast genome. In Vitro exchange studies reveal that when apo-(His6)-β2 (Hisβ2) is mixed with β′2, apo-Hisββ′ forms quantitatively within 2 min. In contrast, holo-ββ′ fails to exchange with apo-Hisβ2 to form holo-Hisββ and β′2. Isolation of genomically encoded tagged β or β′ from yeast extracts gave a 1:1 complex of β and β′, suggesting that ββ′ is the active form. The catalytic activity, protein concentrations, and Y‚ content of the rnr4∆ and wild type (wt) strains were compared to clarify the role of β′ in ViVo. The Y‚ content of rnr4∆ is 15-fold less than that of wt, consistent with the observed low activity of rnr4∆ extracts (