The structurally related diiron centers of methane monooxygenase, ribonucleotide reductase, and fatty acid delta9-desaturase react with O2 to carry out completely different biochemical transformations. The chemistry of their O2 activation reactions will be studied by freeze- trapping reaction intermediates and characterizing their structures by resonance Raman spectroscopy. This technique can specifically identify vibrational modes of Fe-O-Fe groups as well as peroxo, high-valent iron, and free-radical species, particularly when verified by 18/O-isotope shifts. Methane monooxygenase, an enzyme that hydroxylates unactivated C-H bonds, will be investigated for O-O vibrations of a peroxo intermediate and Fe/IV-O vibrations of a high-valent iron intermediate. Ribonucleotide reductase, an enzyme that uses its diiron center and O2 to generate a tyrosyl free radical, will be investigated for Fe-O-Fe vibrations to pinpoint the location of a precursor free radical at the diiron center. Delta9-desaturase, an enzyme that uses O2 to oxidize fatty acid chains, will be probed for the aforementioned intermediates, as well as the binding of NO, an O2 analog. These studies of O2 activation will increase our understanding not only of enzyme mechanisms, but also of mechanisms of oxygen and free-radical toxicity. In contrast, the Cu- S(Cys) sites in cupredoxin-type proteins mediate long-range electron transfer between different metal centers. This site has surprising flexibility in accommodating ligand mutations and may have actually converted to a dinuclear copper cluster in the CuA domain of cytochrome c oxidase. Identification of the coordination geometry in the CuA site will be pursued by observing changes in Raman vibrational frequencies with Cu, S, and N isotopes and correlating these changes with normal coordinate analysis predictions for different structural models. Knowledge of the structure of the unusual CuA site is important to understanding the function of cytochrome oxidase as the terminal electron acceptor in cellular respiration.
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