Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit

Abstract

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides. Eukaryotes have an alpha(2)beta(2) form of RNR consisting of two homodimeric subunits, proteins R1 (alpha(2)) and R2 (beta(2)). The R1 protein is the business end of the enzyme containing the active site and the binding sites for allosteric effectors. The R2 protein is a radical storage device containing an iron center-generated tyrosyl free radical. Previous work has identified an RNR protein in yeast, Rnr4p, which is homologous to other R2 proteins but lacks a number of conserved amino acid residues involved in iron binding. Using highly purified recombinant yeast RNR proteins, we demonstrate that the crucial role of Rnr4p (beta') is to fold correctly and stabilize the radical-storing Rnr2p by forming a stable 1:1 Rnr2p/Rnr4p complex. This complex sediments at 5.6 S as a betabeta' heterodimer in a sucrose gradient. In the presence of Rnr1p, both polypeptides of the Rnr2p/Rnr4p heterodimer cosediment at 9.7 S as expected for an alpha(2)betabeta' heterotetramer, where Rnr4p plays an important role in the interaction between the alpha(2) and the betabeta ' subunits. The specific activity of the Rnr2p complexed with Rnr4p is 2,250 nmol deoxycytidine 5'-diphosphate formed per min per mg, whereas the homodimer of Rnr2p shows no activity. This difference in activity may be a consequence of the different conformations of the inactive homodimeric Rnr2p and the active Rnr4p-bound form, as shown by CD spectroscopy. Taken together, our results show that the Rnr2p/Rnr4p heterodimer is the active form of the yeast RNR small subunit.

Figure 1

SDS/PAGE analyses of purified recombinant yeast RNR subunits (1–2 μg per lane) isolated from E. coli. Lanes 1 and 8, molecular mass markers (kDa); lane 2, Rnr1p; lane 3, Rnr3p; lane 4, H₆Rnr2p; lane 5, Rnr4p; lane 6, H₆Rnr2p/Rnr4p complex; lane 7, H₆Rnr2p/ΔRnr4p complex.

Figure 2

Activity of the yeast small subunit. (A) Specific activity of the H₆Rnr2p/Rnr4p complex (1 μg) in the presence of increasing amounts of Rnr1p. The specific activities were calculated per milligram of H₆Rnr2p in the complex. (B) Activities of 2 μg of the H₆Rnr2p/Rnr4p complex (○, left y axis) and of a mixture of 1 μg of H₆Rnr2p and 1 μg of Rnr4p (●, right y axis), both times with 5 μg of Rnr1p. (C) Specific activities of the H₆Rnr2p/Rnr4p complex (solid bars, right y axis) and of a H₆Rnr2p and Rnr4p mixture (hatched bars, left y axis) in a yeast RNR assay at different temperatures.

Figure 3

CD spectra of H₆Rnr2p (thick line), Rnr4p (thin line), a mixture of H₆Rnr2p and Rnr4p (dashed line) and the H₆Rnr2p/Rnr4p complex (dotted line).

Figure 4

Analyses of the oligomeric structure of yeast RNR by sucrose gradient centrifugation. The numbers correspond to the different fractions, which were around 50 in all experiments. The fractions were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. (A) 240 μg of H₆Rnr2p. (B) 220 μg of Rnr4p. (C) 140 μg of the H₆Rnr2p/Rnr4p complex. (D) 140 μg of the H₆Rnr2p/Rnr4p complex and 480 μg of Rnr1p. (E) 140 μg of the H₆Rnr2p/Rnr4p complex and 480 μg of Rnr1p in the presence of 4 mg/ml of Rnr1p throughout the gradient. (F) Computer assisted quantification of the amounts of H₆Rnr2p (o) and Rnr4p (●) in (E). The ordinate is pixels per mm². In D, E and F, fraction 20 corresponds to 9.7S and fraction 35 corresponds to 5.6S.