Why multiple small subunits (Y2 and Y4) for yeast ribonucleotide reductase? Toward understanding the role of Y4

摘要

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 is directly involved in the reduction, and R2 contains the diferric-tyrosyl radical (Y⋅) cofactor essential for the initiation of reduction. Saccharomyces cerevisiae has two RNRs; Y1 and Y3 correspond to R1, whereas Y2 and Y4 correspond to R2. Y4 is essential for diferric-Y⋅ formation in Y2 from apoY2, Fe²⁺, and O2. The actual function of Y4 is controversial. Y2 and Y4 have been further characterized in an effort to understand their respective roles in nucleotide reduction. (His)6-Y2, Y4, and (His)6-Y4 are homodimers, isolated largely in apo form. Their CD spectra reveal that they are predominantly helical. The concentrations of Y2 and Y4 in vivo are 0.5–2.3 μM, as determined by Western analysis. Incubation of Y2 and Y4 under physiological conditions generates apo Y2Y4 heterodimer, which can form a diferric-Y⋅ when incubated with Fe²⁺ and O2. Holo Y2Y4 heterodimer contains 0.6–0.8 Y⋅ and has a specific activity of 0.8–1.3 μmol⋅min⋅mg. Titration of Y2 with Y4 in the presence of Fe²⁺ and O2 gives maximal activity with one equivalent of Y4 per Y2. Models for the function of Y4 based on these data and the accompanying structure will be discussed.