ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase.
Interactions between subunit a and oligomeric subunit c are essential for the coupling of proton translocation to rotary motion in the ATP synthase. A pair of previously described mutants, R210Q/Q252R and P204T/R210Q/Q252R [L.P. Hatch, G.B. Cox and S.M. Howitt, The essential arginine residue at position 210 in the a subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity, J. Biol. Chem. 270 (1995) 29407-29412] has been constructed and further analyzed. These mutants, in which the essential arginine of subunit a, R210, was switched with a conserved glutamine residue, Q252, are shown here to be capable of both ATP synthesis by oxidative phosphorylation, and ATP-driven proton translocation. In addition, lysine can replace the arginine at position 252 with partial retention of both activities. The pH dependence of ATP-driven proton translocation was determined after purification of mutant enzymes, and reconstitution into liposomes. Proton translocation by the lysine mutant, and to a lesser extent the arginine mutant, dropped off sharply above pH 7.5, consistent with the requirement for a positive charge during function. Finally, the rates of ATP synthesis and of ATP-driven proton translocation were completely inhibited by treatment with DCCD (N,N'-dicyclohexylcarbodiimide), while rates of ATP hydrolysis by the mutants were not significantly affected, indicating that DCCD modification disrupts the F(1)-F(o) interface. The results suggest that minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c.
Ishmukhametov
Robert R
RR
Department of Biological Sciences, Box 750376, Southern Methodist University, Dallas, TX 75275-0376, USA.
Pond
J Blake
JB
Al-Huqail
Asma
A
Galkin
Mikhail A
MA
Vik
Steven B
SB
D025181:ATP Synthetase Complexes / Q000737:chemistry / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D021122:Protein Subunits
D021122:Protein Subunits; D000255:Adenosine Triphosphate; D025181:ATP Synthetase Complexes