BIOENERGETIC MECHANISMS IN MULTICENTER ENZYMES

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The mechanisms by which three metalloenzymes, cytochrome oxidase, methane monooxygenase (MMO), and soluble guanylate (sGC) operate will be studied. Cytochrome oxidase maintains electron-transfer activity in the mitochrondrion by catalyzing the reduction of dioxygen to water. It also contributes directly to the transmembrane proton gradient by pumping protons stoichiometrically with electrons transported to O2, as follows: 4 cyt c2+ +O2+8H+in yields 4cyt c3+ +4H+ out +2H2O. Dioxygen chemistry occurs at the heme alpha3/CuB binuclear center. We and others have proposed that CuB is directly involved in the proton pump. Our recent data also suggests that a unique, histidine cross-linked tyrosyl radical occurs during O2 reduction and is involved in driving proton translocation. By using time-resolved Raman spectroscopy, we intend to measure vibrational spectra for partially metabolized intermediates in O2 reduction and to assess mechanisms by which the protein imposes proton control on the rates of these reactions. Site-directed mutants of sphaeroides oxidase are available, and we will characterize the functional roles of specific amino acids. We intend to use FTIR spectroscopy, magnetic-resonance, computational methods, chemical modeling, and chemical labeling to assess whether redox-active tyrosyl residue is intimately involved in O-O bond cleavage and to test models for ligand exchange at the CuB site that may be relevant to its proposed role in the proton pump. For MMO, we plan to characterize the Compound Q intermediate. Our Raman data indicate that this species, which carries out oxygen-bond insertion chemistry, has an Fe-2+ O2+ diamond cone structure. For sGC, we will study the molecular mechanisms that allow efficient NO/O2 discrimination and begin work aimed at understanding how NO binding triggers cyclase activity.