Characterization of NO adducts of the diiron center in protein R2 of Escherichia coli ribonucleotide reductase and site-directed variants; implications for the O₂ activation mechanism

The R2 subunit of Escherichia coli ribonucleotide reductase contains a diiron site that reacts with O₂ to produce a tyrosine radical (Y122·). In wild-type R2 (R2-wt), the first observable reaction intermediate is a high-valent [Fe^III-Fe^IV] state called compound X, but in related diiron proteins such as methane monooxygenase, Δ⁹-desaturase, and ferritin, peroxodiiron(III) complexes have been characterized. Substitution of iron ligand D84 by E within the active site of R2 allows an intermediate μ-1,2-peroxo)diiron species to accumulate. To investigate the possible involvement of a bridging peroxo species within the O₂ activation sequence of R2-wt, we have characterized the iron-nitrosyl species that form at the diiron sites in R2-wt, R2-D84E, and R2-W48F/ D84E by using vibrational spectroscopy. Previous work has shown that the diiron center in R2-wt binds one NO per iron to form an antiferromagnetically coupled [{FeNO}⁷]₂ center. In the wt and variant proteins, we also observe that both irons bind one NO to form a {FeNO}⁷ dimer where both Fe-N-O units share a common vibrational signature. In the wt protein, v(Fe-NO), δ(Fe-N-O), and v(N-O) bands are observed at 445, 434 and 1742 cm^-1, respectively, while in the variant proteins the v(Fe-NO) and δ(Fe-N-O) bands are observed ∼10 cm^-1 higher and the v(N-O) ∼10 cm^-1 lower at 1735 cm^-1. These results demonstrate that all three proteins accommodate fully symmetric [{FeNO}⁷]₂ species with two identical Fe-N-O units. The formation of equivalent NO adducts in the wt and variant proteins strongly favors the formation of a symmetric bridging peroxo intermediate during the O₂ activation process in R2-wt.