Reaction of NO with the Reduced R2 Protein of Ribonucleotide Reductase from Escherichia colf

The active R2 protein of ribonucleotide reductase from Escherichia coli contains a catalytically essential tyrosine radical at position 122 (Tyr₁₂₂•) that is formed during the reaction of dioxygen with the nearby diiron(II) center. To gain insight into the mode of dioxygen binding, the reaction of the O₂ analog NO with the diiron(II) centers of R2_red has been investigated by spectroscopic methods. R2_red reacts with NO to form an adduct with visible absorption features at 450 and 620 nm and Mossbauer parameters (δ = 0.75 mm/s, ∆E_Q = -2.13 and -1.73 mm/s) typical of those observed for S = 3/2 {FeNO}⁷ complexes of other non-heme iron proteins. However, unlike other non-heme {FeNO}⁷ complexes, this adduct is EPR silent. Our Mossbauer studies show that each iron site of R2_red binds one NO to form local S = 3/2 {FeNO}⁷ centers which then couple antiferromagnetically (J ≈ 5 cm^-1, H = JS1·S2) to afford an {FeNO}₂ center (77% of total iron). This {FeNO}₂ center decomposes with a first-order rate constant of 0.013 min^-1 to form R2_met, accompanied by the release of N₂O. These observations suggest that both iron(II) ions of the two diiron(II) centers of R2_red have available sites for NO binding, in agreement with the crystallographic results on R2_red, and that the bound NO molecules are sufficiently close to each other to permit N-N bond formation to produce N₂O. These observations support the proposal that dioxygen binding may also involve both metal ions of the diiron(II) center to form a (µ-1,1-, or µ-1,2-peroxo)-diiron(III) center. This observed reactivity of R2_red with NO may contribute to the in vivo inhibition of ribonucleotide reductase by NO.