Magnetic circular dichroism spectroscopy as a probe of the geometric and electronic structure of non-heme ferrous enzymes

Abstract

Mononuclear non-heme iron active sites are present in a variety of enzymes involed in a wide range of important biological functions requiring dioxygen. These include superoxide dismutases, oxidases, extra- and intradiol dioxygenases, cis-dihydroxylases, pterin- and α-ketoglutarate-dependent hydroxylases, lipoxygenases, and bleomycin. Both the ferrous and ferric oxidation states are involved in catalysis for different enzymes in this class and substrate-and oxygen-bound intermediates have been observed. Much less is known about the active sites in these enzymes relative to the heme systems as the non-heme iron centers are less spectroscopically accessible, particularly at the ferrous oxidation level. The application of magnetic circular dichroism (MCD) spectroscopy has greatly advanced the level of understanding in these systems as this technique allows the direct observation of the ferrous d→d ligand field transitions, which are generally obscured by solvent and protein vibrations in optical absorption spectroscopy owing to the weak extinction coefficients and are often electron paramagnetic resonance silent due to relatively large ground-state sublevel splittings and fast relaxation times. The energies of the d→d transitions give the splitting of the excited-state e g orbitals which can be correlated with the coordination number and geometry at the ferrous center. In addition, the ground states of high-spin ferrous complexes are described as an s=2 spin manifold which undergoes axial zero-field splitting into M s=±2, ±1, 0 components separated by 3 D and D, respectively. The non-Kramers M s=±2 doublet is lowest in energy for D<0, and is further rhombically split by an amount σ in the absence of a magnetic field. The unusual variable-temperature variable-field MCD saturation behavior observed for such systems can be interpreted by including the effects of σ as well as z-polarization, linear B-terms, and the population of low-lying excited states. The non-degenerate M s=0 state is lowest in energy for D>0 and the resulting MCD saturation behavior can be analyzed by including the effects of off-axis Zeeman terms and z-polarized eletronic transitions. The spin Hamiltonian parameters obtained through analysis of the variable-temperature variable-field MCD saturation data are further interpreted in terms of the ligand field splitting of the ground-state t 2g set of d-orbitals. This MCD methodology has been applied to several biologically relevant mononuclear non-heme ferrous systems to directly probe the active site geometric and electronic structures and to gain mechanistic information about their catalytic cycles. MCD spectroscopy has been used to study the native ferrous active site of phthalate dioxygenase and its interaction with substrate and exogenous ligands, which has previously been difficult to study due to the additional presence of a [2Fe-2S] Rieske cluster. The native form of soybean lipoxygenase exists as a mixture of species in solution that has been defined through the application of CD and MCD spectroscopies. This MCD methodology has also been used to elucidate the nature of the ferrous active site in bleomycin, which represents an important deviation from mononuclear non-heme iron enzymes in that it exhibits low-energy charge transfer transitions and performs chemistry similar to heme systems. The MCD methodology presented in this review has been employed to obtain molecular level insight into the catalytic mechanisms of these important enzyme systems and to understand the differences in active site geometric and electronic structures which relate to differences in oxygen reactivity.