This research examines the physical and cellular biochemistry of a novel exchange protein found in the gram-negative anaerobe Oxalobacter formigenes. In this anaerobe, the exchange of divalent oxalate for monovalent formate forms the basis for generation of proton-motive force. This antiport reaction is unprecedented among "chemiosmotic" carriers for its role in cellular biochemistry and unique among anion exchange proteins for its electrogenic character. Assays have been devised to reconstitute this antiport reaction without loss of function and are used as a guide to purify the exchange protein from membranes of O. formigenes. After the protein is purified experiments will reveal the kinetic reaction mechanism (Ping Pong or Simultaneous) and that portion of the reaction cycle which carries charge over the membrane. Using residue-specific chemical probes, monoclonal antibodies directed against the antiport protein, and the derived amino acid sequence predictions will be made about the physical structure. Oxalobacter formigenes, a gram-negative anaerobe, is a cell whose sole source of metabolic energy derives from the decarboxylation of oxalic acid. It has been established that membranes of O. formigenes contain a carrier that displays the electrogenic exchange of divalent oxalate and monovalent formate. This reaction is of particular interest to cellular biochemistry as a mechanism whereby the organism sustains a proton-motive force. Thus, the antiport of precursor and product results in the net entry of negative charge, while consumption of a proton during oxalate decarboxylation changes internal pH. This is a novel use for such a "secondary" carrier and is of interest to determine whether other cells that utilize carboxylic acids also exploit such antiport reactions. This research will examine the molecular properties of this carrier and its overall function in cellular biochemistry. This work will contribute to the current understanding of how bacteria organize their cellular functions.
