STRUCTURAL STUDIES ON EUKARYOTIC MOCO CONTAINING ENZYMES

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Enzymes containing the molybdenum cofactor catalyze important reactions in the global sulfur, nitrogen and carbon cycles and are currently classified into four different families. Sulfite oxidase and xanthine dehydrogenase are key members of two of these four families. Sulfite oxidase catalyzes the physiologically vital oxidation of sulfite to sulfate and also plays an important role in detoxifying exogenously supplied sulfite and sulfur dioxide. Sulfite oxidase deficiency is a severe disease in humans, which mostly leads to death in early childhood. Recently, the first mutations in the gene encoding sulfite oxidase have been identified. Xanthine dehydrogenase is an important enzyme in catabolic purine metabolism. Xanthine dehydrogenase deficiency known as xanthinuria causes an accumulation of insoluble xanthine, which can lead to acute renal failure in humans. Furthermore, xanthine dehydrogenase after conversion into the oxidase form of the enzyme has been implicated in reperfusion injury. The experiments outlined in this proposal, which comprise a combination of biochemical, biophysical and molecular biology methods, are aimed at a better understanding of both enzymes and their involvement in human diseases. The specific aims of this proposal are divided into five categories as outlined below: (1) Structural characterization of recombinant rat and human sulfite oxidase: The wildtype structures of the rat and human enzymes will be determined. These structures will provide the reference frame for the structural characterization and interpretation of the mutations, which lead to sulfite oxidase deficiency. (2) Sulfite oxidase deficiency: We will structurally characterize the seven so far identified mutants and compare them with the wildtype structures in order to characterize the structural changes, which compromise enzyme activity. (3) Catalytic mechanism of sulfite oxidase: We will identify residues which are involved in catalysis, characterize the Mo coordination geometry at different oxidation states and gain further insight into catalysis by structural characterization of substrate free and inhibitor forms of the enzyme. (4) Electron transfer mechanism: Both, the intra- and intermolecular electron transfer mechanisms will be studied and residues, which are crucial for either pathway will be identified. We will also structurally characterize the complex between sulfite oxidase and its physiological electron acceptor cytochrome c. (5) Crystal structure analysis of bovine xanthine dehydrogenase: Determination of the overall structure as well as characterization of the active site by determination of structures with bound inhibitors and determination of the NAD+ bound form.