Reaction Mechanism of Ribonucleoside Diphosphate Reductase from Escherichia coli

Ribonucleoside diphosphate reductase consists of two nonidentical subunits, proteins B1 and B2. The enzyme catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The electrons required in this reduction are transported from NADPH via a flavoprotein, thioredoxin reductase, to a low molecular weight protein, thioredoxin. The reduced form of thioredoxin acts as hydrogen donor in ribonucleotide reduction. Both thioredoxin and thioredoxin reductase contain oxidation-reduction active disulfides participating as electron carriers during catalysis. In this paper, data are presented which show that in the absence of hydrogen donor, ribonucleotide reductase reduces a limited amount of ribonucleotides at the expense of sulfhydryls of protein B1. A maximal value of 3 moles of deoxyribonucleotide was obtained per mole of B1 and at the same time 6 moles of sulfhydryls were oxidized. The reaction requires the presence of protein B2. The same initial rate of cytidine diphosphate reduction was obtained in the presence and in the absence of reduced thioredoxin. Electron transfer occurred readily between fully reduced B1 and the oxidation-reduction active disulfide of thioredoxin. Steady state kinetics of ribonucleotide reductase indicated that the enzyme acted by a ping-pong mechanism, i.e. alternated between two stable forms during catalysis. Based on these results it is proposed that in the reduction of ribonucleotides, electrons flow from thioredoxin to oxidationreduction active disulfides of protein B1. The dithiols formed interact by unknown mechanisms with a free radical species in protein B2 (Atkin, C. L., Thelander, L., Reichard, P., and Lang, G. (1973) J. Biol. Chem. 248, 7464–7472) to reduce the ribonucleotide.