The objective of this proposal is to define the kinetic mechanisms and physiological roles of a variety of oxidoreductases and electron carrier proteins found in methylotrophic and autotrophic bacteria; and to exploit these bacteria as systems for the study of the molecular mechanisms of protein biosynthesis, export, and assembly. These soluble enzymes and proteins possess a variety of interesting redox centers including flavins, iron-sulfur centers, hemes, copper, and the novel pyrroloquinoline quinone (PQQ) cofactor; many are inducible and synthesized to very high levels; and many are exported to and function in the periplasmic space of these bacteria. The structural and physical properties of these redox proteins will be determined and the kinetics of the interactions of redox proteins which function in sequence will be analyzed. This will allow specific inferences to be drawn and tested concerning structure-function relationships which pertain to the mechanisms of intramolecular and intermolecular electron transport. The role of PQQ in catalysis by methylamine dehydrogenase will be defined. This will have broad significance in that the mammalian copper-containing amine oxidases, which play an important role in regulating the levels of biogenic amines, also possess PQQ. Each of the redox proteins to be studied is representative of families of proteins which are ubiquitous in nature. As such, the structural and functional analogies between these bacterial proteins and their eukaryotic counterparts will be characterized by physical, immunological, and kinetic analyses. In vitro translation and translocation systems will be developed to study the mechanisms by which the expression of these respiratory proteins are regulated, precursor proteins are processed, prosthetic groups are attached to apoproteins, multisubunit enzymes are assembled, and polypeptides are transported across biological membranes. This information should greatly enhance our understanding of the biogenesis of proteins which are involved in energy transduction and of exported proteins in general. As energy metabolism is vital to all living things, the proposed studies will be relevant to our understanding of a critical biological process.
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