This project is devoted to the study of transition-metal ions in protein active sites, particularly those involved in oxygen activation and electron transfer. The major focus is on ribonucleotide reductase (RRase), a key enzyme in DNA biosynthesis and a target for chemotherapy. The enzymatically active form of RRase contains a stable tyrosyl radical and an Fe-O-Fe moiety, both of which are generated during the reaction of the diferrous protein with O2. The chemistry of the reactivation reaction will be proved by resonance Raman spectroscopy, a technique that can specifically identify vibrational modes of tyrosyl radicals, Fe-O-Fe groups, and peroxo and ferryl intermediates. Diferrous RRase will be reconstituted with isotopically labelled O2 to show whether the mu-oxo group is derived from solvent or dioxygen and, thus, yield information about a possible ferryl intermediate. Site-directed mutants of RRase will reveal the effect of individual Fe ligands on Fe-O-Fe cluster geometry and reactivity towards O2. Studies of the well-characterized RRase from E.coli will be extended to mammalian-type RRase, an enzyme of greater clinical importance. The presence of an Fe-O-Fe site will be verified, and the stability of the tyrosyl radical will be determined. The accessibility of the diiron center to small molecules will be probed by measuring the rates of oxo-bridge exchange and H2O2 binding. Such information is important to understanding the mechanism of inactivation of RRase by hydroxyurea, a chemotherapeutic agent. A second focus is on cupredoxin-type proteins and enzymes in which a conserved blue copper center mediates long-range electron transfer between different proteins or between different metal sites within the same protein. Azurin and pseudoazurin mutants with amino acid changes at or near the blue copper site will be examined by RR spectroscopy to see how these changes affect the structures of the cysteine and histidine ligands and associated ligand-binding loops. Nitrite reductase reactions with azide and cyanide will be investigated by IR and Raman spectroscopy to determine whether nitrite reduction occurs at the blue copper site, the non-blue copper site, or even an alternate enzyme active site.
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