Abstract
The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b(562), the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the epsilon subunit of the enzyme. According to the concept of rotational catalysis, because epsilon is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme. E. coli cells expressing the cytochrome b(562) fusion in place of wild-type epsilon grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation. Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate. Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly. These results provide initial evidence supporting rotational catalysis in vivo. In membrane vesicles, the cytochrome b(562) fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping. Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping. Thus, all fusions to epsilon generated an uncoupled component of ATPase activity. These results imply that a function of the C terminus of epsilon in F(1)F(0) is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP. Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of gammaepsilon in the inappropriate direction after ATP is bound at the catalytic site. It is proposed that the C-terminal domain of epsilon functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound.
Highlights
ATP synthase is the enzyme responsible for the production of ATP during oxidative phosphorylation
Plasmid Construction and Protein Purification—To investigate subunit rotation in ATP synthase in vivo, a number of proteins of increasing mass were fused to the C terminus of ⑀
In Vivo Evidence Supporting the Rotational Mechanism of ATP Synthase—It is believed that the ATP synthesis/hydrolysis function of the peripheral F1 sector is coupled to the movement of protons through the F0 sector by the rotation of the ␥⑀c10–14 complex relative to the remainder of the enzyme
Summary
General Methods—Recombinant DNA techniques were performed as described by Sambrook et al [21]. The PCR product was inserted into the NdeI and AvrII sites of pSD132 to produce pSD143, a plasmid containing the cytochrome b562 gene fused to the C terminus of ⑀ through a Gly-Ser linker. To facilitate the cloning of the ⑀ fusion proteins into the unc operon, the 238-bp SgfI/PstI fragment of pSD132 was moved into pACWU1.2 using the same sites to produce plasmid pSD135 This plasmid contains the entire unc operon with the ⑀A137H mutation encoding the NdeI site and the AvrII site after the stop codon of the uncC gene. E-Orange ⑀ (⑀ fused to flavodoxin through a 20-residue linker) was precipitated with 40 –55% saturated ammonium sulfate, applied to a DEAE-Sepharose column, and eluted with a linear 0 – 600 mM gradient of NaCl. The resulting fractions were pooled and subjected to gel filtration on a Sephacryl S-200 column. The establishment of a proton gradient in membrane vesicles in response to ATP was monitored essentially as described by Jiang et al [38], using an ISA Fluorolog 3-11 spectrofluorimeter
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