Azide Binding to the Diferrous Clusters of the R2 Protein of Ribonucleotide Reductase from Escherichia coli

Ribonucleotide reductases provide the only biosynthetic pathway for the generation of deoxyribonuc1eotides.l The Escherichia coli enzyme is a 1 : 1 complex of R1 and R2 proteins;2 the X-ray structure of the latter establishes the presence of two nonheme (p-oxo)diiron(III) clusters in close proximity to Tyr 122,3 which must be oxidized to its radical form to elicit enzyme activity.435 It is the reaction of the differrous form (R2,d) with 0 2 that generates the tyrosyl radical.6~~ However, the coordination environment of the diiron clusters in R2,d is less well characterized than that of the diferric form (R2met). It is clear that the strong antiferromagnetic coupling of the diferric clusters4J is significantly decreased upon reduction, but there is some disagreement on the nature of the metal-metal interaction.a The N M R resonance for the imidazole N-H protons of the coordinated histidines shifted from 24 ppm in R2,,, to 57 ppm in R2,d; furthermore, the temperature dependence of the 57-ppm peak suggested a weak antiferromagnetic coupling of ca. lO(10) cm-l ( H = JS1S2).7 On the other hand, preliminary EPR studies on R2,d in our laboratory revealed the presence of a low-field EPR signal arising from a ground state;6 such signals are usually associated with an integer spin system, which in this case might be associated with a ferromagnetically coupled diferrous center. More recently, a multifield saturation magnetization study of R2,d found J = -0.6(4) cm-1.8 Since fitting the temperature dependence of an Fe(I1)-shifted N M R signal requires a number of simplifying assumptions and the bulk susceptiblity measurements assumes a homogeneous sample, we have taken advantage of the higher resolution of the EPR method to reconcile these conflicting observations. We have used integer spin EPR spectroscopy with azide as a probe to examine the magnetic properties of R2,d and its complexes with azide and report here the first spectroscopic evidence of exogenous ligand binding to the diferrous clusters of the R2 protein. The EPR spectrum of R2,d9 with HIJJH (Figure 1) exhibits a signal a t g = 14.410 whose temperature dependence indicates that it arises from a ground-state doublet. This, together with the signal position and line shape, indicates that the signal originates