REACTION OF NO WITH THE REDUCED R2 PROTEIN OF RIBONUCLEOTIDE REDUCTASE FROM ESCHERICHIA-COLI

The active R2 protein of ribonucleotide reductase from Escherichia coli contains a catalytically essential tyrosine radical at position 122 (Tyr(122)(.)) 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 O-2 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 (delta = 0.75 mm/s, Delta 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 approximate to 5 cm(-1), H = JS(1) . S-2) 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 (mu-1,1-, or mu-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.