Structural studies of two redox protein systems involved in the oxidation of amines will be carried out. One of the systems consists of an iron-sulfur flavoprotein, trimethylamine dehydrogenase (TMADH), which undergoes intramolecular electron transfer from the flavin to the iron-sulfur center during catalysis, and an electron transferring flavoprotein (ETF), the specific the electron acceptor for TMADH. The other system, yeast copper amine oxidase (YAO), is a quinoprotein. It contains topaquinone (TPQ), a catalytic cofactor derived from a gene-encoded tyrosine side chain and a copper atom. The structure of TMADH is known at 1.7 A resolution and several mutants have been prepared and characterized. The questions addressed in these studies are the formation and role of the covalent link to FMN, the specificity and stabilization of substrate binding at the active site, the mechanism of substrate oxidation, control of intramolecular electron transfer kinetics and the nature of the intermolecular electron transfer step to ETF. The structures of several of these mutants will be determined and additional mutants will be designed and structurally characterized. The native structure will be extended in resolution to 1.0 A from flash frozen crystals using synchrotron radiation and the structures of several functional derivatives, aimed at the intramolecular electron transfer step, will be completed. The structure of ETF will be completed and used to model the intermolecular electron transfer complex with TMADH. The structure of YAO has recently been determined by molecular replacement and single isomorphous replacement. The refined structure shows the orientation of TPQ and of the copper center in the active configuration of the resting enzyme. The structures of several functional derivatives of YAO and of several mutants designed to probe its biogenic and catalytic properties will be determined. The questions addressed by these studies are the biosynthesis of TPQ, the roles of certain conserved residues near the TPQ and elsewhere, substrate specificity, structure and function at the active site, and access of the active site to substrate. The structure will also be used to design additional mutants which will also be structurally analyzed.
Showing the most recent 10 out of 25 publications
