Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli

The enzyme activity of Escherichia coli ribonucleotide reductase requires t he presence of a stable tyrosyl free radical and diiron center in its small er R2 component. The iron/radical site is formed in a reconstitution reacti on between ferrous iron and molecular oxygen in the protein. The reaction i s known to proceed via a paramagnetic intermediate X, formally a Fe-III-Fe- IV state. me have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e . a long-living X-like intermediate and a transient tyrosyl radical, were o bserved only with azide and under none of the other conditions, A crystal s tructure of the mutant protein R2 E238A/Y122F with a diferrous iron site co mplexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coo rdination from Glu-204, A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinat e Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.