CATALASE AND PHOTOSYNTHETIC WATER OXIDASE

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The long term objective of this proposal is to better understand enzyme catalysis by metalloenzymes which utilize metal clusters. Specifically, the molecular structure and mechanism of catalysis by two multinuclear manganese enzymes which appear to share structural and functional homologies will be compared: manganese catalase and the photosynthetic water oxidizing enzyme. The photosynthetic water oxidizing enzyme is a complex multisubunit protein which utilizes a tetramanganese cluster and calcium and chloride ions to catalyze the photochemical oxidation of water to oxygen by an unknown mechanism. Catalysis of this chemistry is so complex that only a single type of enzymatic site has been identified in nature, from the most primitive cyanobacteria to present day plants. The health of all aerobic organisms, including man, is critically dependent upon this process since it is the only renewable source of atmospheric oxygen in the biosphere. Manganese catalases are rare in comparison to heme catalases and possess a spin-coupled dimanganese center as the catalytic site in place of the simpler heme cofactor. Like the heme catalases they function to remove the cellular by-product hydrogen peroxide which is a common and potent toxin to all cells. The mechanism of this process is not fully understood. The enzyme from the high temperature thermophile, Thermus thermophilus, will be studied. The proposed methods and their specific goals are: 1) High resolution multinuclear (1H, 15N, 17O, 19F, 35C1) electron nuclear double resonance (ENDOR) spectroscopy will be used to identify the magnetic nuclei located within magnetic contact (< 5A) to the manganese ions in several oxidation states: The ligand hyperfine data will be used to determine the atomic positions using a newly developed model. 2) High frequency 55Mn ENDOR and 35 GHz electron paramagnetic resonance (EPR) spectroscopies will be used to measure the nuclear electric quadrupole interaction and the magnetic hyperfine interaction of the individual Mn ions which monitor the internal electric and magnetic fields surrounding the cluster. This will be used to determine how this changes upon binding of inhibitors, inorganic cofactors, and substrate and upon redox changes of the Mn ions; 3) The identity of the protein derived radical produced in the calcium depleted water oxidase will be investigated by ENDOR spectroscopy; 4) Synthesis of catalytically active structural models of manganese catalase and their characterization by kinetic and spectroscopic methods; collaborative studies of synthetic dimanganese complexes with Dr. N. Kitajima which are structural models for multinuclear Mn enzymes; 5) Collaborative resonance Raman studies with Dr. R. Czernuszewicz on manganese catalase and synthetic mimics to determine the active site structure of magnetically silent oxidation states.