The objectives of this proposal are (i) to define the mechanisms of catalysis and electron transfer by selected quinoprotein dehydrogenases and their quinone prosthetic groups, (ii) to define the mechanisms of long range electron transfer from these dehydrogenases to soluble redox proteins which subsequently transfer electrons to the respiratory chain and (iii) to define the factors which stabilize the specific interactions between these soluble weakly-associating proteins that facilitate inter- molecular electron transfer. These processes will be examined by steady- state and rapid kinetics, direct binding assays, thermodynamic analysis and collaborative biophysical and x-ray crystallographic studies. Quinoproteins are a class of bacterial and eukaryotic enzymes which possess novel quinones as prosthetic groups and which catalyze the oxidation of biologically relevant amines, alcohols, sugars and aldehydes. Quinoprotein dehydrogenases are distinct from most other oxidoreductases in that their immediate electron acceptors are not oxygen or soluble cofactors, such as NAD+, but are electron transferring proteins, such as cytochromes and copper proteins. Quinone cofactors, such as pyrroloquinoline quinone [PQQ], have been shown to be essential nutrients for mammals and have been implicated as possible therapeutic agents for a variety of disorders related to oxidative stress. As such, an understanding of the role of such quinones in catalysis and electron transfer will be of broad significance. The proposed studies will describe the roles of the enzyme-bound quinones in the oxidation of amines and alcohols by selected quinoprotein dehydrogenases. This will be of general interest as we have previously shown that the mechanism used by methylamine dehydrogenases to oxidize amines is very similar to that used by the mammalian amine oxidizing quinoproteins, plasma amine oxidase and lysyl oxidase. Elucidation of the factors which influence the specific protein-protein interactions between quinoproteins and their electron acceptor proteins, and facilitate intermolecular electron transfer, will further our understanding of the process of protein- protein recognition which is common to a wide range of biological phenomena. Characterization of the mechanisms and pathways of long range intermolecular electron transfer will allow us to better understand the fundamental process of respiration at the molecular level.
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