Crystal structure of reduced protein R2 of ribonucleotide reductase: the structural basis for oxygen activation at a dinuclear iron site

Abstract

Background Ribonucleotide reductases (RNRs) catalyze the formation of the deoxyribonucleotides that are essential for DNA synthesis. The R2 subunit of Escherichia coli RNR is a homodimer containing one dinuclear iron centre per monomer. A tyrosyl radical is essential for catalysis, and is formed via a reaction in which the reduced, diferrous form of the iron centre activates dioxygen. To help understand the mechanism of oxygen activation, we examined the structure of the diferrous form of R2. Results The crystal structures of reduced forms of both wild type R2 and a mutant of R2 (Ser211→ Ala) have been determined at 1.7 å and 2.2 å resolution, respectively. The diferrous iron centre was compared to the previously determined structure of the oxidized, diferric form of R2. In both forms of R2 the iron centre is coordinated by the same carboxylate dominated ligand sphere, but in the reduced form there are clear conformational changes in three of the carboxylate ligands and the bridging μ-oxo group and two water molecules are lost. In the reduced form of R2 the coordination number decreases from six to four for both ferrous ions, explaining their high reactivity towards dioxygen. The structure of the mutant Ser211→ Ala, known to have impaired reduction kinetics, shows a large conformational change in one of the neighbouring helices although the iron coordination is very similar to the wild type protein. Conclusions Carboxylate shifts are often important for carboxylate coordinated metal clusters; they allow the metals to achieve different coordination modes in redox reactions. In the case of reduced R2 these carboxylate shifts allow the formation of accessible reaction sites for dioxygen. The Ser211→ Ala mutant displays a conformational change in the helix containing the mutation, explaining its altered reduction kinetics.