The Structural and Functional Role of the y-e Rotor in Escherichia Coli F0F1 ATP Synthase

The --c subunits of the Escherichia coli F O F 1 ATP synthase make up the rotor assembly, which couples proton transport to ATP catalysis. Previous EPR and functional studies from our laboratory suggest that interactions in the - and -c interfaces play important roles in efficient coupling. To further investigate the structure and dynamics of the interface, we have used the site-directed spin labeling strategy of EPR spectroscopy to define the structure of  subunit in the - -c subunit interface. EPR spectra of isolated  subunits with spin labels at single-cysteine substitutions from E29C to A44C show an alternating mobility indicating a -strand secondary structure. When bound to F 1 complex, spectra of spin-labeled P40C-T43C show large decreases of mobility, which are likely due to tertiary contacts with the  subunit. The L42C mutant subunit binds to the F 1 complex with lower affinity indicating that the hydrophobic interaction contributes significant amount of binding energy in - interface. The EPR spectra of spin labeled 29, 33, 35, 36, 37 and 39 subunit-F 1 complexes demonstrate large mobility decreases when F 1 is reconstituted with the membranous F O sector, suggesting that these residues directly interact with subunit c. Single-cysteine mutations introduced in the -c interface do not disrupt energy coupling between F O and F 1 , implying that the -c interface is the main channel for the energy coupling mechanism and one role of  is to strengthen the -c interaction and ii coordinate conformations of  and c subunits for optimal coupling efficiency. Deuterium-Hydrogen exchange and X-ray footprinting methods were used to explore the solvent-accessibility changes of the rotor and rotor-stator interface regions in the presence and absence of ATP. However, due to the problem of deuterium-back-exchange, solvent accessibility map of the - rotor could not be established based on the deuterium-hydrogen exchange method. From X-ray footprinting analysis, the nucleotide-dependent changes of oxidative rates of Met178 and Met379 suggest the possible important contacts between  and  3  3 hexamer during rotation. Decreases of oxidative rates of Met15, Met49, and Phe61 in the presence of ATP provide evidence for the nucleotidedependent movement of C-terminal helices of  subunit.

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