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The R2 subunit of Escherichia coli ribonucleotide reductase (RNR) contains a cofactor which consists of a stable tyrosyl radical ('Y 122) adjacent to a p-oxo-bridged diiron(II1) cluster. This cofactor assembles spontaneously when apo R2 is treated with Fez+ and 0 2 . By using rapid kinetic techniques, two kinetically competent intermediates in this assembly process were recently identified (Bollinger, J. M., Jr. et al. Science 1991,253,292-298). One of the intermediates is a paramagnetic iron species. By using the rapid freeze-quench technique and a mutant R2 subunit in which Y122 is replaced with F, 1 equiv of this intermediate per R2 subunit can be trapped. Previous electron paramagnetic resonance (EPR) and MBssbauer studies suggested that this species is a spin coupled system involving two high-spin ferric ions and a free radical (a "diferric radical species") (Bollinger, J. M., Jr. et al. J. Am. Chem. SOC. 1991, I 1 3,6289-629 1). In the present study, M6ssbauer spectra have been recorded over a wide range of applied fields (60 mT-8 T), and detailed analysis of the MBssbauer data consolidates the initial finding. The observed isomer shifts (0.55 mm/s for iron site 1 and 0.36 mm/s for site 2) and quadrupole splittings (-1 mm/s) are typical for high-spin ferric species. The magnetic hyperfine coupling tensors, A, for the two iron sites are relatively isotropic (within 20%), a feature which is unique to high-spin Fe(II1). By using a simple three-spin coupling model (SI = 5/2 and S2 = 5/2 for the two iron sites, and SJ = '/2 for the radical), both the signs and magnitudes of the observed A values for the iron sites (Al/g,& = -52.5 T and A2/gn/3,, = +24 T) can be explained with an intrinsic A value that is consistent with high-spin Fe(II1). The asymmetry in the A values for the two iron sites is a result of the spin orientations: the spin of the radical is parallel to that of the negative-A-value Fe site and antiparallel to that of the positive-A-value Fe site. Since it has been suggested on the basis of functional analogy with heme-iron-dependent peroxidases that generation of *Y 122 in RNR-R2 may involve a high-valent iron-oxo intermediate, the possibility that this paramagnetic iron species contains Fe(1V) has also been carefully examined. Both the EPR and the MBssbauer data are incompatible with such a possibility. IntroductionRibonucleotide reductases (RNRs) catayze the first committed and rate limiting step of DNA biosynthesis, the reduction of nucleotides to 2'-deoxyn~cleotides.1~ The RNRs from E. coli and mammals, as well as those encoded by several medically important viruses (e.g. herpes simplex, and rabies), share a common quaternary structural motif and metallocofactor requirement. Each is composed of two homodimeric subunits, which are both required for catalytic activity. The larger subunit, designated R1, contains the following in each of its protomers:(1) binding sites for the nucleoside diphosphate substrates and the nucleoside and deoxynucleoside triphosphate allosteric effectors and (2) multiple...

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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 R2red has been investigated by spectroscopic methods. R2red reacts with NO to form an adduct with visible absorption features at 450 and 620 nm and Mössbauer parameters (delta = 0.75 mm/s, delta EQ = -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 Mössbauer studies show that each iron site of R2red binds one NO to form local S = 3/2 [FeNO]7 centers which then couple antiferromagnetically (J approximately 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 R2met, accompanied by the release of N2O. These observations suggest that both iron(II) ions of the two diiron(II) centers of R2red have available sites for NO binding, in agreement with the crystallographic results on R2red, 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 R2red with NO may contribute to the in vivo inhibition of ribonucleotide reductase by NO.

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