Electron transfer associated with oxygen activation in the B2 protein of ribonucleotide reductase from Escherichia coli

Each of the two β peptides which comprise the B2 protein of Escherichia coli ribonucleotide reductase (RRB2) possesses a nonheme dinuclear iron cluster and a tyrosine residue at position 122. The oxidized form of the protein contains all high spin ferric iron and 1.0-1.4 tyrosyl radicals per RRB2 protein. In order to define the stoichiometry of in vitro dioxygen reduction catalyzed by fully reduced RRB2 we have quantified the reactants and products in the aerobic addition of Fe(II) to metal-free RRB2_apo utilizing an oxygraph to quantify oxygen consumption, electron paramagnetic resonance to measure tyrosine radical generation, and Mössbauer spectroscopy to determine the extent of iron oxidation. Our data indicate that 3.1 Fe(II) and 0.8 Tyr¹²² are oxidized per mol of O₂ reduced. Mössbauer experiments indicate that less than 8% of the iron is bound as mononuclear high spin Fe(III). Further, the aerobic addition of substoichiometric amounts of ⁵⁷Fe to RRB2_apo consistently produces dinuclear clusters, rather than mononuclear Fe(III) species, providing the first direct spectroscopic evidence for the preferential formation of the dinuclear units at the active site. These stoichiometry studies were extended to include the phenylalanine mutant protein (Y122F)RRB2 and show that 3.9 mol-equivalents of Fe(II) are oxidized per mol of O₂ consumed. Our stoichiometry data has led us to propose a model for dioxygen activation catalyzed by RRB2 which invokes electron transfer between iron clusters.