Enzymic and chemical reduction of the iron center of the Escherichia coli ribonucleotide reductase protein R2. The role of the C-terminus.

Abstract

The active form of protein R2, the small subunit of ribonucleotide reductase, contains a diferric center and a free radical localized at Tyr122. Hydroxyurea scavenges this radical but leaves the iron center intact. The resulting metR2 protein is inactive. The introduction of a radical into metR2 is dependent on the reduction of the iron center. In Escherichia coli, this is achieved by an enzyme system consisting of a NAD(P)H:flavin oxidoreductase and a poorly defined protein fraction, fraction b. Assuming that the iron center is deeply buried within the protein, electron transfer is suggested to occur over long distances. Site-directed mutagenesis allowed us to identify two invariant residues, Tyr356 at the C-terminal part of the protein and Tyr122 located 0.5 nm away from the closest iron atom, as mediators of this electron transfer. We also found that deazaflavins were excellent catalysts in the photoreduction of the iron center of metR2 and generation of the tyrosyl radical, providing the simplest and most efficient model for the physiological flavin reductase/fraction b activating system. The properties of the model reaction are described.