Defining the active form of ribonucleotide reductase from Saccharomyces cerevisiae in vitro and in vivo

Abstract

Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides. Saccharomyces cerevisiae RNR is a class I RNR composed of a dimeric large subunit (RI), containing the active site and allosteric effector sites, and a dimeric small subunit (R2) which houses the diferric tyrosyl radical (Y.) cofactor required for RNR activity. S. cerevisiae has two Rl-like proteins, Y1 and Y3 and two R2-like proteins, Y2 and Y4. In vitro studies have focused on defining the active form of the R2 subunit. As isolated, Y2 and Y4 are homodimers and neither can bind iron to generate the ... cofactor. When mixed, the Y2 and Y4 homodimers rapidly form a heterodimer that can be activated with iron to form the ... cofactor located in the Y2 subunit. While the apo-Y2Y4 protein is capable of undergoing slow exchange of its protomers, loading of iron into the heterodimer prevents this slow reorganization. To define the role of Y4 in vivo, the concentrations of Y2 and ... in the wt and Y4 deletion strain have been determined. Thought deletion of Y4 leads to a >15 fold increase in the concentration of Y2, the concentration of ... is at least 15 fold less than the amount observed in the wt yeast strain. We have concluded form this work and from our work in collaboration with Prof. Amy Rosenzweig's laboratory in which the structures of Y2, Y4 and the heterodimer were determined that Y4 plays an important role in assembly of the diiron cofactor in Y2 through stabilization of a local conformation of Y2 allowing for iron binding and that the active form of yeast R2 is a Y2Y4 heterodimer. These conclusions are supported by in vivo experiments.

(cont.) Using a yeast strain containing an N-terminally tagged Y2 under control of the native promoter, a soluble, active Y2Y4 heterodimer is isolated. This heterodimer contains 0.45±0.08 ..., 1.45±0.05 irons and a specific activity of 2.3±0..5 !Rmolmin' -mg'l. Wt and several deletion strains, including Smll[delta], Rnrl[delta], Rnr4[delta], and Crt 1[delta] strains, have been characterized by Western blotting to quantitate the concentrations of the RNR subunits, by EPR spectroscopy to quantitate the concentration of ... cofactor in vivo and by activity assays with yeast extracts to measure the total amount of RNR activity. These results demonstrate that in vivo every Y2Y4 heterodimer has a diferric-... cofactor and that the activity of Y1 limits RNR activity in vivo. Furthermore, the information acquired has allowed us better understanding how the protein concentration, cofactor generation and subunit localization regulate RNR activity in vivo.