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Research was conducted to examine the geometric and electronic structure of the reduced iron center. The methodologies used were applied circular dichroism, magnetic circular dichroism, variable-temperature-variable-field molecular circular dichroism, X-ray absorption pre-edge and extended X-ray absorption fine structure spectroscopies. Results indicate that the coordination number increases upon reduction which is attributed to the coordination of a second solvent ligand.

The energy, intensity, and splitting of the 1s -> 3d pre-edge feature and its sensitivity to the electronic and geometric structure of the iron site was measured. This was based on the XAS Fe-K edge data on high- and low-spin ferrous and ferric inorganic model complexes. Results reveal that the energy splitting and intensity distribution of the pre-edge features of these complexes vary with spin state, oxidation state, geometry and bridging ligation.

Magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies were utilized to determine the structure of the catalytically relevant ferrous active site of phenylalanine hydroxylase (PAH). MCD and XAS analysis of PAH indicated the presence of a resting ferrous site with a six-coordinate distorted octahedral geometry. The resting catalytically active ferrous site of PAH also contains longer bond distances compared to small molecule complexes.

The high-spin ferrous complexes of bleomycin (Fe(II)BLM) and the structural analog PMAH, Fe(II)PMA +, support a dissociative mechanism for oxygen reactivity. PMAH is a macrocyclic ligand with deprotonated amide, pyrimidine, imidazole and secondary and primary amine functionalities. An effective iron (II)-to-pyrimidine backbonding occurs in Fe(II)BLM and Fe(II)PMA + due to the presence of low energy charge transfer transition states. A low spin state of both ferrous complexes is obtained when they coordinate to pi-acceptor exogenous ligands, yielding long-lived oxygen intermediates.

A study of ferrous soybean lipoxygenase-1 (SLO-1) using near-infrared circular and magnetic circular dichroism and variable temperature, variable-field MCD shows that SLO-1 exists as a mixture of 40% five- and 60% six-coordinate forms. The two crystal structures of SLO-1 contain three histidines and a carboxylate terminus isoleucine. Experimental conditions do not have any significant effect on the five and six coordinate mixture of SLO-1. X-ray absorption iron K-edge and pre-edge data indicates that mammalian and recombinant human 15-LOs and SLO-1 in glycerol exist as six coordinates.

An integrated approach GNXAS enables the effective analysis of extended X-ray absorption fine structure (EXAFS) spectroscopic data. The GNXAS methodology evaluates structural parameters of molecular systems based on the study of multiple-scattering effects in the EXAFS data. Application of this approach to the iron complexes, Fe(acac)3, Na(Fe(OH2)EDTA) and K3Fe(CN)6 (acac = acetylacetonate and EDTA = ethylenediaminetetraacetic acid), of known structures shows its reliability and advantages. A detailed analysis of Fe-C-N unit of K3Fe(CN)6 using GNXAS reveals the ability of this method to accurately determine bond angles above 150 degrees.

X-ray absorption spectroscopy (XAS) enables the study of the oxidation and spin state of iron in activated Bleomycin (BLM). Activated BLM possesses a six-coordinate low-spin ferric complex and has no short Fe-O bond. Its oxygen activated intermediate has a structure similar to peroxy-ferric complexes, which are expected to play an important role in the oxygen activation of heme enzymes.

Spin restricted and spin unrestricted SCF-X alpha-SW electronic structure calculations on two S = 3 2 iron-nitrosyl complexes indicate an antiferromagnetic coupling between high spin Fe3+ and NO- S = 1. Spectral studies support the antiferromagnetic coupling. Strong donor binding interaction of NO- with the Fe3+ forms a highly covalent Fe3+-NO(-) bond. Magnetic circular dichroism experiments reveal that electronic structures of these complexes are similar to those of the nitrosyl derivative of the non-heme iron site in soybean lipoxygenase. Spectral data of these nitrosyl complexes help determine bonding differences in oxygen intermediates of various non-heme iron proteins.

EXAFS and a new theoretical data analysis package, GNXAS, help determine Fe-N-O angle in various (FeNO)7 complexes. They determine the bent or linear nature of Fe-N-O unit and provide more angular information for small molecular transition metal complexes having complex coordination environments. They also determine the coordination geometry of NO and O2 in metalloprotein active sites.

Soybeans lipoxygenase nitric oxide (SBL-NO) and a pair of S = 3/2 ground-state model complexes FeL(NO)(N3)2 were analyzed using spectroscopic and theoretical approaches. A novel bonding description of the S = 3/2 (FeNO)+ unit is antiferromagnetically coupled to NO-(S = 1) through straightforward orbital overlap. The electronic structure of the nitrosyl complex of SBL resembles that of the (FeNO)+ model systems.

Studies pre-edge features of iron complexes using X-ray absorption spectroscopy. Types of ligating atoms; Relative intensities of electric quadrupole transitions; Characterization of iron active sites.