Iron and Free Radical in Ribonucleotide Reductase

Abstract Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits (Protein B1 and Protein B2), both required for activity. Protein B2 contains 2 iron atoms and a lesser, variable amount of a paramagnetic species, characterized by g = 2.0047 EPR and 410 nm electronic absorptions. Except for the 410 nm band, Protein B2 much resembles oxy- and methemerythrins in electronic spectra. The paramagnetic species is probably organic free radical rather than metal, is dependent upon the presence of iron in Protein B2, is directly correlated with enzymatic activity, and is destroyed by NH2OH and hydroxyurea (Ehrenberg, A., and Reichard, P. (1972) J. Biol. Chem. 247, 3485–3488). We here describe complete removal of iron from Protein B2, reconstitution to active metalloenzyme, and Mossbauer spectroscopy of 57FeProtein B2. Addition of excess Fe2+ to metal-free protein yielded reconstituted/reactivated protein with 2:1 iron:subunit stoichiometry and a hemerythrinlike electronic spectrum, but with 120 to 390% of the original specific enzymatic activity and correspondingly increased 410 nm and EPR absorptions. NH2OH- or hydroxyurea-inactivated protein was also converted to highly active enzyme subunit by the same iron substitution procedures. Mossbauer and electronic spectra suggested that Protein B2 contains 2 nonidentical high spin Fe(III) ions in an antiferromagnetically coupled binuclear complex that resembles both methydroxohemerythrin and oxyhemerythrin. Below 195°K the iron in Protein B2 was completely diamagnetic, in confirmation of the previous assignment of the 410 nm- and EPR-absorbing species as stable organic free radical of unknown structure. We propose that the function of iron in Protein B2 is the initial generation of radical from a protein-bound group, and that stability of the radical depends upon some continuing interaction with the iron center. The radical is apparently less stable than is metalloprotein structure: this would account for the variability of radical content in native protein and for the g 100% yield of radical obtainable by substitution of the iron. We further propose that the free radical participates in the reduction of ribonucleotides by the enzymatically active Protein B1-Protein B2 complex.