We have used 8-azidoadenosine 5-triphosphate (8-N 3 ATP) to investigate the nucleotide-binding sites on the NrdD subunit of the anaerobic ribonucleotide reductase from T4 phage. Saturation studies revealed two saturable sites for this photoaffinity analog of ATP. One site exhibited half-maximal saturation at approximately 5 M [␥-32 P]8-N 3 ATP, whereas the other site required 45 M. To localize the sites of photoinsertion, photolabeled peptides from tryptic and chymotryptic digests were isolated by immobilized Al 3؉ affinity chromatography and high performance liquid chromatography and subjected to amino acid sequence and mass spectrometric analyses. The molecular masses of the photolabeled products of cyanogen bromide cleavage were estimated using tricine-SDS-polyacrylamide gel electrophoresis. Overlapping sequence analysis localized the higher affinity site to the region corresponding to residues 289 -291 and the other site to the region corresponding to residues 147-160. Site-directed mutagenesis of Cys 290 , a residue conserved in all known class III reductases, resulted in a protein that exhibited less than 10% of wild type enzymatic activity. These observations indicate that Cys 290 may reside in or near the active site. High performance liquid chromatography analysis revealed that photoinsertion of [␥-32 P]8-N 3 ATP into the site corresponding to residues 147-160 was almost completely abolished when 100 M dATP, dGTP, or dTTP was included in the photolabeling reaction mixture, whereas 100 M ATP, GTP, CTP, or dCTP had virtually no effect. Based on these nucleotide binding properties, we conclude that this site is an allosteric site analogous to the one that has been shown to regulate substrate specificity of other ribonucleotide reductases. There was no evidence for a second allosteric nucleotide-binding site as observed in the anaerobic ribonucleotide reductase from Escherichia coli.Ribonucleotide reductases catalyze the reduction of ribonucleotides to their corresponding 2Ј-deoxyribonucleotides. Currently, they are divided into three classes based on differences in cofactor requirements, structural composition, and type of radical employed for catalysis (1, 2). Because of the importance of maintaining a balanced supply of deoxyribonucleotides for DNA synthesis (3, 4), they are enzymes that are subject to complex allosteric regulation (5, 6). Although there may be striking differences in primary sequence, the same general mechanism for allosteric regulation appears to apply to all ribonucleotide reductases described to date, with only subtle differences observed (7). Three different nucleotide-binding sites have been localized on the prototypical class I reductase from Escherichia coli using photoaffinity labeling (8) and x-ray crystallography (9, 10). Two of these sites are allosteric sites that coordinate the reduction of all four ribonucleotide substrates at a single active site. One allosteric site binds only ATP and dATP and regulates the overall activity of the enzyme. The other allosteric site, via i...