Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides. Three classes have been identified, all using free-radical chemistry but based on different cofactors. Classes I and II have been shown to be evolutionarily related, whereas the origin of anaerobic class III has remained elusive. The structure of a class III enzyme suggests a common origin for the three classes but shows differences in the active site that can be understood on the basis of the radical-initiation system and source of reductive electrons, as well as a unique protein glycyl radical site. A possible evolutionary relationship between early deoxyribonucleotide metabolism and primary anaerobic metabolism is suggested.

The anaerobic ribonucleotide reductase from Escherichia coli contains a glycyl radical as part of its polypeptide structure. The radical is generated by an enzyme system present in E. coli. The reductase is coded for by the nrdD gene located at 96 min. Immediately downstream, we now find an open reading frame with the potential to code for a 17.5-kDa protein with sequence homology to a protein required for the generation of the glycyl radical of pyruvate formate lyase. The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase. The "activase" contains iron, required for activity. The general requirements for generation of a glycyl radical are identical for the reductase and pyruvate formate lyase. For the reductase, the requirement of an iron-containing activase suggests the possibility that the iron-sulfur cluster of the enzyme is not involved in radical generation but may participate directly in the reduction of the ribonucleotide.

The subtle differences in base size and hydrogen bonding pattern at the effector site are communicated to major conformational changes in the active site. We propose that the altered overlap of Phe-194 with the substrate base governs hydrogen bonding patterns with main and side chain hydrogen bonding groups in the active site. The relevance for evolution is discussed.

It has been recently recognized that the class III anaerobic ribonucleotide reductase requires the presence of a second activating gene product, NrdG. We have proposed that the role for NrdG involves the generation of an oxygen sensitive glycyl free radical within the NrdD enzyme. In this article we present the generation of such a glycyl free radical within the T4 NrdD subunit and its dependence upon the phage NrdG subunit. Initially, an overexpression system was created that allowed the joint production of T4 NrdD and T4 NrdG. With this system and in the presence of T4 NrdG, an oxygen-sensitive cleavage of NrdD was observed that mimicked the cleavage observed in phage infected Escherichia coli extracts. Under anaerobic conditions the presence of T4 NrdD with NrdG revealed a strong doublet EPR signal (g ‫؍‬ 2.0039). Isotope labeling of the NrdD with [ H]glycine and [13 C]glycine, respectively, confirmed the presence of a stabilized glycine radical. The unpaired electron is strongly coupled to C-2 in glycine and the doublet splitting originates from one of the ␣-protons. The glycine residue at position 580 was determined to be the radical containing residue through sitedirected mutagenesis studies involving a G580A NrdD mutant. The glycyl radical generation was specific for the T4 NrdG, and the host E. coli NrdG was found to be unable to activate the phage reductase. Finally, anaerobic purification revealed the holoenzyme complex to contain iron, whereas the NrdD polypeptide was found to lack the metal. Our results suggest a tetrameric structure for the T4 anaerobic ribonucleotide reductase containing one homodimer each of NrdD and NrdG, with a single glycyl radical present.

Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4. By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism. Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se. We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys-564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical. These cysteines make up a CX 2 C-CX 2 C motif in the vicinity of the stable radical at Gly-580. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation.