This project is devoted to the investigation of structural properties of metalloprotein active sites, particularly the elucidation of the interactions between the metal ion and its ligands and neighboring groups. In the previous grant period, we established that vibrational modes of metal ion clusters are very sensitive to hydrogen bonding. In oxyhemerythrin, the bound dioxygen molecule exists as a hydroperoxide ion hydrogen-bonded to the oxo-bridged binuclear iron center. We plan to extend these studies to the important enzyme, ribonucleotide reductase, which uses a hemerythrin-related iron center to control a tyrosyl free radical near the active site, and is mandatory for the activity of the enzyme. Site-directed mutants are available that will permit detailed spectroscopic investigations of the active site of the reductases. Binuclear iron centers are also implicated in purple acid phosphatase and as nucleation sites in core formation in ferritin. We plan to use resonance Raman spectroscopy to characterize the presence of these Fe-O-Fe clusters in these proteins. Despite the similarity in the iron cores of the binuclear iron proteins, their spectroscopic behavior has been shown to be quite variable. Model studies will establish the relationship between the structural properties and nature of ligands to account for this diversity. The crystal structure of hemocyanin shows that no amino acid side chains bridge the copper atoms; yet they are magnetically coupled. We plan to investigate hemocyanin for evidence of the presence of any solvent-derived bridging groups between the copper atoms. In addition, the importance of hydrogen bonding among metal clusters, ligands, and neighboring amino acids is becoming increasingly apparent, and vibrational spectroscopy has shown that it is sensitive to H-bonding from isotope exchange experiments. We plan to look for stabilization of the bound dioxygen in hemocyanin via H-bonding effects. In ferredoxins, crystal data have established that both the bridging sulfur atoms and the cysteinate sulfurs are involved in extensive H-bonding interactions with the protein. We plan to explore whether such interactions can be measured by resonance Raman spectroscopy. Spectroscopic studies of Type 2 copper enzymes will be enhanced by derivatization with copper binding ligands that give strong LMCT bands. Oxygenated intermediates of the heme-containing peroxidases will be investigated and identified by their characteristic vibrational spectra.
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