Subunit a of F 1 F 0 ATP synthase is required in the H ؉ transport driven rotation of the c-ring of F 0 , the rotation of which is coupled to ATP synthesis in F 1 . The three-dimensional structure of subunit a is unknown. In this study, Cys substitutions were introduced into two different transmembrane helices (TMHs) of subunit a, and the proximity of the thiol side chains was tested via attempted oxidative cross-linking to form the disulfide bond. Pairs of Cys substitutions were made in TMHs 2/3, 2/4, 2/5, 3/4, 3/5, and 4/5. Cu ؉2 -catalyzed oxidation led to cross-link formation between Cys pairs L120C(TMH2) and S144C(TMH3), L120C(TMH2) and G218C(TMH4), L120C(TMH2) and H245C(TMH5), L120C(TMH2) and I246C(TMH5), N148C(TMH3) and E219C(TMH4), N148C(TMH3) and H245C(TMH5), and G218C(TMH4) and I248C(TMH5). Iodine, but not Cu ؉2 , was found to catalyze cross-link formation between D119C(TMH2) and G218C(TMH4). The results suggest that TMHs 2, 3, 4, and 5 form a four-helix bundle with one set of key functional residues in TMH4 (Ser-206, Arg-210, and Asn-214) located at the periphery facing subunit c. Other key residues in TMHs 2, 4, and 5, which were concluded previously to compose a possible aqueous access pathway from the periplasm, were found to locate to the inside of the four-helix bundle.The H ϩ -transporting F 1 F 0 ATP synthases of oxidative phosphorylation utilize the energy of a transmembrane electrochemical gradient of protons or Na ϩ to mechanically drive the synthesis of ATP via two coupled rotary motors in the F 1 and F 0 sectors of the enzyme (1, 2). In the intact enzyme, ATP synthesis or hydrolysis takes place in the F 1 sector at the surface of the membrane, synthesis being coupled to H ϩ transport through the transmembrane F 0 sector. In Escherichia coli and other bacteria, F 1 consists of five subunits in an ␣ 3  3 ␥ 1 ␦ 1 ⑀ 1 stoichiometry (3). High resolution structures of the homologous ␣ 3  3 ␥ subunits of the bovine mitochondrial enzyme revealed two extended ␣-helices of the ␥ subunit extending through the core of a hexamer of alternating ␣ and  subunits (4, 5). Rotation of the asymmetric ␥ subunit was proposed to alternately close and open the catalytic site within the  subunit to force synthesis and release of ATP according to the binding change mechanism (4, 6). In a series of pioneering experiments, ATP hydrolysis and synthesis were subsequently shown to be coupled to the rotation of the ␥ subunit (7-9).The F 0 sector of bacterial ATP synthase spans the inner or plasma membrane. In E. coli and Bacillus subtilis PS3, F 0 is composed of three subunits in a likely ratio of a 1 b 2 c 10 (2, 10, 11). Subunit c spans the membrane as a hairpin of two ␣-helices and is packed in a ring or cylinder-like structure with the first transmembrane helix (TMH) 2 on the inside and the second TMH on the outside (12, 13). In E. coli, Asp-61 at the center of the second TMH is thought to undergo protonation and deprotonation as each subunit of the oligomeric ring moves past a stationary subunit a. In the Na ϩ t...
The genes of Salmonella enterica serovar Typhimurium LT2 encoding functions needed for the utilization of tricarballylate as a carbon and energy source were identified and their locations in the chromosome were established. Three of the tricarballylate utilization (tcu) genes, tcuABC, are organized as an operon; a fourth gene, tcuR, is located immediately 5 to the tcuABC operon. The tcuABC operon and tcuR gene share the same direction of transcription but are independently transcribed. The tcuRABC genes are missing in the Escherichia coli K-12 chromosome. The tcuR gene is proposed to encode a regulatory protein needed for the expression of tcuABC. The tcuC gene is proposed to encode an integral membrane protein whose role is to transport tricarballylate across the cell membrane. tcuC function was sufficient to allow E. coli K-12 to grow on citrate (a tricarballylate analog) but not to allow growth of this bacterium on tricarballylate. E. coli K-12 carrying a plasmid with wild-type alleles of tcuABC grew on tricarballylate, suggesting that the functions of the TcuABC proteins were the only ones unique to S. enterica needed to catabolize tricarballylate. Analyses of the predicted amino acid sequences of the TcuAB proteins suggest that TcuA is a flavoprotein, and TcuB is likely anchored to the cell membrane and probably contains one or more Fe-S centers. The TcuB protein is proposed to work in concert with TcuA to oxidize tricarballylate to cis-aconitate, which is further catabolized via the Krebs cycle. The glyoxylate shunt is not required for growth of S. enterica on tricarballylate. A model for tricarballylate catabolism in S. enterica is proposed.Tricarballylate (1,2,3-propanetricarboxylate) is structurally related to citric acid; hence, its catabolism is thought to proceed via the tricarboxylic acid (Krebs) cycle (Fig. 1). Tricarballylate is the causative agent of grass tetany, a disease in ruminants characterized by a pronounced magnesium ion deficiency. Reports in the literature have established a strong correlation between high levels of trans-aconitate (trans-1-propene-1,2,3-tricaboxylate) in the rumen with the incidence of the disease (6). In the rumen, trans-aconitate is rapidly converted to tricarballylate in a single reductive step (Fig. 2) (25). It is not clear how tricarballylate causes grass tetany; however, it is known that tricarballylate is a good chelator of magnesium ions and that it enhances excretion of this important divalent metal ion (33). In addition, tricarballylate is known to be a potent inhibitor of aconitase, a key enzyme of the Krebs cycle (26,29,33). This effect on aconitase makes tricarballylate particularly toxic to organisms that rely on the Krebs cycle for energy generation and are unable to catabolize tricarballylate into nontoxic metabolites.In the rumen, Selenomonas ruminantium is the primary producer of tricarballylate (25). Not surprisingly, this bacterium has been the target of studies aimed at blocking the accumulation of this compound. The bacterium Acidaminococcus ferm...
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