Current mechanistic models of the FoFj ATP synthase predict that H+ transport in FO drives rotation of the f-f dimer relative to the catalytic 3/3 hexamer in FI. Rotation of the dimer, in turn, drives cooperative catalysis with all three catalytic sites participating. To probe the role of interactions between subunits in the rotor, random mutations were introduced into a region of the E. coli 7 subunit believed to interact with Fj subunit t and several FO subunit c (Watts, S.D., Tang, C., and Capaldi, R.A. (1996) J. Biol Chem. 271, 283412H347). One mutation, 7E208K, was of particular interest because it caused A temperature-sensitive defect in oxidative phosphorylation-dependent growth. In membrane vesicles from the 7E208K-strain, ATP hydrolytic rates increased with temperature while ATP-dependent H+ pumping decreased with temperatures above 28°C even though the membranes did not become leaky to protons. Arrhenius analysis of steady-state ATP hydrolysis of the 7E208K mutant FoF] enzyme revealed that the temperature dependent uncoupling coincided with a significant increase in the activation energies suggesting that additional bonds must be broken to achieve the catalytic transition state. Significantly, the 7E280K FI dissociated from FO had catalytic turnover and thermodynamic parameters similar to the wild-type enzyme. These results suggest that the 7E208K mutation perturbs the interaction between the FI portion of the rotor (7 and f subunits) and the FO portion (c subunits) in a manner that renders tho enzyme uncoupled. Supported bv PHS grant GM50957-.
|Published - 1998
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- General Biochemistry, Genetics and Molecular Biology
- Cell Biology