Circular dichroism and magnetic circular dichroism studies of the fully reduced binuclear non-heme iron active site in the Escherichia coli R2 subunit of ribonucleoside diphosphate reductase

A combination of circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies has been used to probe the geometric and electronic structure of the binuclear Fe(II) active site of the R2 subunit of reduced ribonucleoside diphosphate reductase (R2 RDPR). Excited state data provide the numbers and energies of d --> d transitions which are used to estimate the geometry of each iron atom. Variable-temperature variable-field (VTVH) MCD data are analyzed by using a non-Kramers doublet model to obtain the zero-field splitting (ZFS) and g(11) value of the ground state and the excited state sublevel energies. These results are further interpreted in terms of a spin Hamiltonian which includes the ZFS of each Fe(II) atom combined with the exchange coupling, J, between iron centers. The fully reduced R2 active site is best described as one five- and one four-coordinate Fe(II) atom. The ferrous atoms are weakly antiferromagnetically coupled, J approximate to -0.5 cm(-1), and have opposite ZFS values consistent with iron atoms in different coordination environments. Azide binding studies indicate that reduced R2 has two binding constants for azide which were determined to be 21 +/- 4 M(-1) and 3 +/- 1 M(-1). The active site of the one-azide-bound R2 complex consists of one four- and one six-coordinate Fe(II) atom. VTVH MCD data show the ferrous centers to be antiferromagnetically coupled, J approximate to -2.5 cm(-1). These results suggest that azide binding alters both the geometric and electronic structure of the reduced R2 ground state. High excess azide further perturbs the binuclear ferrous active site and leads to the formation of two distinct two-azide-bound R2 complexes. One component is ferromagnetically coupled, J approximate to 1.0 cm(-1), and is associated with a five- and six-coordinate biferrous center. The second component is weakly coupled, -2.0 < J < 2.0 cm(-1), and consists of one four- and one six-coordinate Fe(II) atom. The azide binding studies are compared to parallel results for deoxyhemerythrin (deoxyHr) and the hydroxylase component of methane monooxygenase (MMOH), and differences are correlated to differences in the bridging ligation of the binuclear ferrous active site.