CIRCULAR-DICHROISM AND MAGNETIC CIRCULAR-DICHROISM STUDIES OF THE FULLY REDUCED BINUCLEAR NONHEME IRON ACTIVE-SITE IN THE ESCHERICHIA-COLI R2 SUBUNIT OF RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE

A combination of circular dichroism (CD) and magnetic circular dichroi sm (MCD) spectroscopies has been used to probe the geometric and elect ronic structure of the binuclear Fe(II) active site of the R2 subunit of reduced ribonucleoside diphosphate reductase (R2 RDPR). Excited sta te data provide the numbers and energies of d --> d transitions which are used to estimate the geometry of each iron atom. Variable-temperat ure variable-field (VTVH) MCD data are analyzed by using a non-Kramers doublet model to obtain the zero-field splitting (ZFS) and g(11) valu e of the ground state and the excited state sublevel energies. These r esults are further interpreted in terms of a spin Hamiltonian which in cludes the ZFS of each Fe(II) atom combined with the exchange coupling , J, between iron centers. The fully reduced R2 active site is best de scribed 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 dif ferent coordination environments. Azide binding studies indicate that reduced R2 has two binding constants for azide which were determined t o be 21 +/- 4 M(-1) and 3 +/- 1 M(-1). The active site of the one-azid e-bound R2 complex consists of one four- and one six-coordinate Fe(II) atom. VTVH MCD data show the ferrous centers to be antiferromagnetica lly coupled, J approximate to -2.5 cm(-1). These results suggest that azide binding alters both the geometric and electronic structure of th e reduced R2 ground state. High excess azide further perturbs the binu clear ferrous active site and leads to the formation of two distinct t wo-azide-bound R2 complexes. One component is ferromagnetically couple d, 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-coordinat e Fe(II) atom. The azide binding studies are compared to parallel resu lts for deoxyhemerythrin (deoxyHr) and the hydroxylase component of me thane monooxygenase (MMOH), and differences are correlated to differen ces in the bridging ligation of the binuclear ferrous active site.