We constructed a single cysteine panel encompassing transmembrane helix two (TM2) of OxlT, the oxalate/ formate antiporter of Oxalobacter formigenes. Among the 21 positions targeted, cysteine substitution identified one (phenylalanine 59) as essential to OxlT expression and three (glutamine 56, glutamine 66, and serine 69) as potentially critical to OxlT function. By probing membranes with a bulky hydrophilic probe (Oregon Green maleimide) we also located a central inaccessible core of at least eight residues in length, extending from leucine 61 to glycine 68. Functional assays based on reconstitution of crude detergent extracts showed that of single cysteine mutants within the TM2 core only the Q63C variant was substantially (>95%) inhibited by thiol-specific agents (carboxyethyl methanethiosulfonate and ethylsulfonate methanethiosulfonate). Subsequent analytical work using the purified Q63C protein showed that inhibition by ethylsulfonate methanethio The antiporter OxlT 1 carries out the electrogenic exchange of divalent oxalate with monovalent formate, a reaction that underlies generation of the proton-motive force in the Gramnegative anaerobe Oxalobacter formigenes (1-3). Although this aspect of bacterial cell biology merits further attention, current studies of OxlT are directed to the development of structural models following the success of electron crystallography, which has established a two-dimensional projection map for this protein (4). Such work may have wider significance because OxlT belongs to the major facilitator superfamily (5), the largest group of evolutionarily related antiporters, uniporters, and symporters (6).The two-dimensional projection map of OxlT reveals a single central cavity representing the substrate translocation pathway (4), but it is not yet possible to recognize the individual helices that border this pathway or to determine which among them contain substrate-binding elements. To address these issues two experimental strategies have been developed. On the one hand, helix proximity is being examined by disulfide trapping in double cysteine variants (7). In addition and as reported here, selected helices are being subjected to biochemical tests to identify a domain(s) that lines the transport pathway (8 -10).Of the twelve OxlT transmembrane helices, TM2 and TM11 are the least hydrophobic (11, 12) and therefore the most likely to specify residues that interact with oxalate (the hydrophilic substrate). In this respect, TM11 has been an attractive candidate for some time because it contains lysine 355, the only charged residue in the OxlT hydrophobic sector and a likely substrate-binding element. Recent work now confirms that TM11 lines the transport pathway and that a positive charge at position 355 is essential to OxlT function (9). By contrast, evidence suggesting that TM2 might line the OxlT pathway has been speculative, deriving largely from its unusually high content of polar residues (Fig. 1) because these may facilitate substrate binding via hydrogen bonding (11).The expe...