Active Site of Ribonucleoside Diphosphate Reductase from Escherichia coli

Ribonucleoside diphosphate reductase is an allosteric enzyme consisting of two nonidentical subunits, proteins Bl and B2. Bl contains dithiols which participate in the oxidation-reduction reactions of electron transport, while B2 contains a free radical essential for activity. Ribonucleoside diphosphates are bound to Bl but not to B2. Addition of 2’-deoxy-2’-chloro ribonucleoside diphosphates to ribonucleotide reductase irreversibly inactivates Bl without affecting B2. The reaction is specific since (a) it requires the presence of active B2, (b) it is controlled by allosteric effecters, (c) Bl is protected against inactivation by the normal substrates, and (d) the chloro-substituted nucleoside monophosphates have no effect. The inactivation of Bl is caused by a modification of the oxidation-reduction dithiols. The chloro derivatives decompose into free base, chloride ion, and 2-deoxyribose 5-diphosphate as a consequence of reaction with the enzyme. 2’-Deoxy-2’-azido ribonucleoside diphosphates cause an irreversible inactivation of B2 without affecting BI. The reaction is specific by the above criteria, indicating that the azido derivatives also bind to the active site of the enzyme. On reduction by ribonucleotide reductase, the azido derivatives function as radical scavengers and selectively destroy the free radical of B2, indicating that this radical participates in ribonucleotide reduction directly. On the basis of these results, a model of the active site of ribonucleotide reductase is proposed in which the site is formed from both Bl and B2. In the site the electron-donating oxidation-reduction active dithiols of Bl are in close contact with the free radical of B2. The azido derivative also inactivates the adenosylcobalamin-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii and a ribonucleotide reductase preparation from calf thymus, indicating a general involvement of free radical intermediates in enzyme-catalyzed ribonucleotide reduction.