SPECTROSCOPIC STUDIES OF THE NONHEME FERRIC ACTIVE-SITE IN SOYBEAN LIPOXYGENASE - MAGNETIC CIRCULAR-DICHROISM AS A PROBE OF ELECTRONIC AND GEOMETRIC STRUCTURE - LIGAND-FIELD ORIGIN OF ZERO-FIELD SPLITTING

Ferric active sites are found in non-heme, non-iron-sulfur enzymes performing a variety of biological functions often involving dioxygen activation. Soybean lipoxygenase (SBL) is an important member of this class catalyzing the hydroperoxidation of unsaturated lipids. The purpose of this study is to use the combination of EPR and variable temperature variable field magnetic circular dichroism (MCD) to probe the ground and excited states in high-spin non-heme ferric enzymes. Low-temperature MCD is used to observe the low-energy spin-forbidden ligand-field excited states that are not obscured by higher energy charge transfer (CT) transitions. Parallel studies on ferric model complexes and predictions of C-term signs and transition energies are presented which enable the ferric geometry to be estimated from these ligand-field spectral features. Saturation magnetization of the MCD bands associated with CT transitions provides polarization information and thus the orientation of the ligand-Fe(+3) bonds relative to the g tensor of the ground doublet which is determined by the zero-field-splitting (ZFS) tensor. Variable-temperature EPR and MCD are used to obtain the ground-state ZFS which is analyzed in terms of the ligand field at the Fe(+3) site. Here we extend our earlier treatment of distorted tetrahedral complexes (Deaton, J. C.; Gebhard, M. S.; Koch, S. A.; Millar, M.; Solomon, E. I. J. Am. Chem. Soc. 1988, 110, 624 1) to distorted octahedral and five-coordinate complexes. Together the saturation magnetization MCD and ZFS analyses enable one to obtain the orientation of specific chromophoric ligands at the active site. This is used to probe both endogenous and exogenous ligand to Fe(+3) CT transitions and provides significant geometric information. Studies of Fe(+3)-SBL show that oxidation of the octahedral Fe(+2) site produces little change in the coordination sphere of the iron center, which likely has at least two histidine ligands in a cis configuration which define the equatorial plane. OCN- and N3- bind to the ferric active site producing rhombic EPR signals which parallel those observed upon hydroperoxide product addition to ferric enzyme. The MCD studies of the associated CT transitions define exogenous ligand interactions with the ferric SBL active site which are of relevance to the catalytic mechanism.