Spectroscopic Studies of the Coupled Binuclear Non-Heme Iron Active Site in the Fully Reduced Hydroxylase Component of Methane Monooxygenase: Comparison to Deoxy and Deoxy-Azide Hemerythrin

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 reduced hydroxylase component of methane monooxygenase (MMOH). Excited-state data provide the numbers and energies of d → d transitions which are interpreted in terms of ligand field calculations to estimate the geometry of each iron. 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_║ value of the ground state and the excited sublevel energies. These results are further interpreted in terms of a spin Hamiltonian which includes the ZFS of each Fe²⁺ combined with the exchange coupling between iron centers. The reduced MMOH contains two five-coordinate ferrous centers with different geometries. VTVH MCD data show the ferrous centers to be ferromagnetically coupled with J ~ 0.3–0.5 cm⁻¹ for the reduced hydroxylase. This indicates that in contrast to deoxyHr which has a binuclear Fe²⁺ site that is antiferromagnetically coupled through a hydroxide bridge, fully reduced MMOH may have a water bridge. The addition of anions, substrates, and inhibitors to reduced MMOH results in no change in the CD spectrum suggesting that these molecules do not bind to the iron or cause large perturbations in the iron site. In contrast, addition of component B causes dramatic changes in the CD and MCD spectra which indicate that one iron in the biferrous active site is altered. Two ferromagnetically coupled Fe(II) centers with distorted five-coordinate square-pyramidal geometries are also found for the MMOH-component B complex. Geometric and electronic structural changes resulting from the addition of component B to reduced MMOH are described and correlated with enhanced reactivity. The above results are compared to parallel results for deoxyHr, and differences are correlated to the difference in dioxygen reactivity (binding versus activation).