Mononuclear non-heme iron active sites are present in a wide range of enzymes involved in a variety of important biological functions requiring dioxygen. These include the lipoxygenases (fatty acid metabolism and regulation of prostaglandin synthesis), extra and intradiol dioxygenases (tyrosine and tryptophan metabolism and degradation of aromatic rings), bleomycin (anticancer drug involved in DNA cleavage), tetrahydroprotein dependent hydroxylases (phenylalanine metabolism related to mental disorders), alpha-ketoglutarate dependent dioxygenases (collagen synthesis), monooxygenases (hydrocarbon and fatty acid hydroxylation), isopenicillin N synthase and superoxide dismutase. Much less is known about the active sites in these enzymes relative to heme systems as these are far less spectroscopically accessible, particularly the high spin ferrous sites which are often not detectable by EPR and do not exhibit intense charge transfer transitions in the visible spectrum. Our research is directed toward developing an excited state spectroscopic approach for the study of non-heme iron enzymes. Absorption, CD, low temperature Magnetic Circular Dichroism (MCD) and resonance Raman spectroscopies are used to study the excited states and variable temperature variable field MCD of an excited state is used to probe the ground state sublevel splittings even in high spin ferrous sites which do not exhibit EPR signals. The data is analyzed in terms of ligand field theory which defines the splitting of the d orbitals as a probe of the electronic and geometric structure of the iron center, and Xalpha-scattered wave molecular orbital calculations of the ligand-to-metal charge transfer transitions which probe the electronic structure associated with substrate and small molecule binding to the iron center. Spectral studies are directed toward the non-heme ferrous and ferric sites, catalytic intermediates and NO complexes of the ferrous sites which serve as stable analogues of reactive small molecule binding. Initial studies have now demonstrated that this excited state spectral approach can provide important information on the active sites in each of these forms of the enzymes. The goals of these studies are to define the geometric and electronic structure of the iron center, its interaction with small molecules and substrate, and the effects of substrate on small molecule binding in that for many non-heme iron enzymes substrate binding enhances dioxygen reactivity. These spectral studies on non-heme iron enzymes should provide molecular level insight into their catalytic mechanisms, define geometric and electronic structure differences which relate to differences in reactivity over the different enzymes in this class, and allow a correlation to heme which in some cases appear to have parallel reactivity.
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