SPECTROSCOPIC STUDIES OF THE COUPLED BINUCLEAR NONHEME 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 dichroi sm (MCD) spectroscopies has been used to probe the geometric and elect ronic structure of the binuclear Fe(II) active site of the reduced hyd roxylase component of methane monooxygenase (MMOH). Excited-state data provide the numbers and energies of d --> d transitions which are int erpreted in terms of ligand field calculations to estimate the geometr y of each iron. Variable-temperature variable-field (VTVH) MCD data ar e analyzed by using a non-Kramers doublet model to obtain the zero fie ld splitting (ZFS) and g(parallel-to) value of the ground state and th e excited sublevel energies. These results are further interpreted in terms of a spin Hamiltonian which includes the ZFS of each Fe2+ combin ed with the exchange coupling between iron centers. The reduced MMOH c ontains two five-coordinate ferrous centers with different geometries. VTVH MCD data show the ferrous centers to be ferromagnetically couple d with J approximately 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, full y reduced MMOH may have a water bridge. The addition of anions, substr ates, and inhibitors to reduced MMOH results in no change in the CD sp ectrum suggesting that these molecules do not bind to the iron or caus e large perturbations in the iron site. In contrast, addition of compo nent B causes dramatic changes in the CD and MCD spectra which indicat e that one iron in the biferrous active site is altered. Two ferromagn etically 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 additio n of component B to reduced MMOH are described and correlated with enh anced reactivity. The above results are compared to parallel results f or deoxyHr, and differences are correlated to the difference in dioxyg en reactivity (binding versus activation).