BACTERIOPHAGE-T4 ANAEROBIC RIBONUCLEOTIDE REDUCTASE CONTAINS A STABLEGLYCYL RADICAL AT POSITION-580

It has been recently recognized that the class III anaerobic ribonucle otide reductase requires the presence of a second activating gene prod uct, NrdG, me have proposed that the rob for NrdG involves the generat ion of an oxygen sensitive glycyl free radical within the NrdD enzyme, In this article we present the generation of such a glycyl free radic al within the T4 NrdD subunit and its dependence upon the phage NrdG s ubunit, Initially, an overexpression system was created that allowed t he joint production of T4 NrdD and T4 NrdG, With this system and in th e presence of T4 NrdG, an oxygen-sensitive cleavage of NrdD was observ ed that mimicked the cleavage observed in phage infected Escherichia c oli extracts, Under anaerobic conditions the presence of T4 NrdD with NrdG revealed a strong doublet EPR signal (g = 2.0039), Isotope labeli ng of the NrdD with [H-2]glycine and [C-13]glycine, respectively, conf irmed the presence of a stabilized glycine radical, The unpaired elect ron is strongly coupled to C-2 in glycine and the doublet splitting or iginates from one of the alpha-protons, The glycine residue at positio n 580 was determined to be the radical containing residue through site -directed mutagenesis studies involving a G580A NrdD mutant, The glycy l radical generation was specific for the T4 NrdG, and the host E. col i NrdG was found to be unable to activate the phage reductase, Finally , anaerobic purification revealed the holoenzyme complex to contain ir on, whereas the NrdD polypeptide was found to lack the metal, Our resu lts suggest a tetrameric structure for the T4 anaerobic ribonucleotide reductase containing one homodimer each of NrdD and NrdG, with a sing le glycyl radical present.