Reaction Mechanism of Ribonucleoside Diphosphate Reductase from Escherichia coli

SUMMARY Ribonucleoside diphosphate reductase consists of two nonidentical subunits, proteins Bl and B2. The enzyme catalyzes the reduction of ribonucleotides to the correspond- ing 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 thiore- doxin 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 sulf- hydryls of protein Bl. A maximal value of 3 moles of deoxyribonucleotide was obtained per mole of Bl and at the same time 6 moles of sulfhydryls were oxidized. The reaction requires the presence of protein B2. The same initial rate of @dine diphosphate reduction was obtained in the presence and in the absence of reduced thioredoxin. Electron transfer occurred readily between fully reduced Bl and the oxidation-reduction active disulfide of thioredoxin. Steady state kinetics of ribonucleotide reductase indicated that the enzyme acted by a ping-pong mechanism,