Spectroscopic Studies on Ferrous Non-Heme Iron Active Sites: Magnetic Circular Dichroism of Mononuclear Fe Sites in Superoxide Dismutase and Lipoxygenase

The geometric and electronic structures of ferrous active sites in Fe superoxide dismutase (FeSD) and soybean lipoxygenase (SBL) have been probed by a combination of optical absorption, circular dichroism, and magnetic circular dichroism spectroscopies in the near-IR spectral region. Distinct ligand Field excited state spectra have been observed for the two ferrous proteins, indicating significant differences in active site structures. Temperature and magnetic field dependent MCD intensity for the excited state spectral features have been used to obtain detailed information on the EPR inaccessible ground states in the ferrous complexes. Analysis of the MCD data has provided estimates of ground-state splitting parameters on the two proteins and two ferrous model complexes; the origin of the unusual field dependence of the MCD intensity at saturation has been explained in terms of Zeeman mixing within a M_s = ±2 non-Kramers doublet which is rhombically split. It is determined that a spin Hamiltonian is not an appropriate description for high-spin ferrous ground-state zero-field splittings for a distorted octahedral or square-pyramidal site. An alternative calculation which includes significant orbital contributions to the ground-state splittings gives the rhombic and Zeeman splittings of the lowest doublet of a ferrous site in terms of spin-orbit coupling within the ligand field split t_2g orbitals. This permits quantitative estimates of the t_2gd orbital splittings from the MCD intensity data. The ground- and excited-state d orbital splittings have been interpreted in terms of geometric and electronic structures for the active site ferrous complexes: for FeSD, a five-coordinate structure and an effective distorted square pyramidal electronic symmetry is indicated by the experimental data, while for SBL a rhombically distorted roughly octahedral six-coordinate structure is determined. Development of ground- and excited-state probes of the ferrous environments in the enzyme active sites has allowed the interaction of the ferrous sites with small molecules to be studied spectroscopically. The ferrous sites in these two proteins appear to be inaccessible to exogenous ligands, which is significant with respect to their O₂ reactivity in catalysis.