The essential carboxyl group in subunit c of the F1F0 ATP synthase can be moved and H(+)-translocating function retained.
The proteolipid subunit c of F1F0-type H(+)-transporting ATP synthases [ATP phosphohydrolase (H(+)-transporting), EC 3.6.1.34] contains a conserved Asp/Glu residue that is thought to function in H+ translocation. To test the importance of the position of this residue in the Escherichia coli enzyme, we used oligonucleotide-directed mutagenesis to move the carboxyl side chain from position 61 to position 58, 60, or 62. Mutant cells with these changes were incapable of growth via oxidative phosphorylation on succinate. An Asp-61----Glu mutant grew on succinate but at 50% the efficiency of wild type. Hence, even minor changes in the position of the carboxyl group can significantly reduce function. In a second approach, slow-growing revertants to an Asp-61----Gly mutant were isolated. In one such revertant, Ala-24 was changed to Asp, while the original Asp-61----Gly mutation remained unchanged. The Asp-24-Gly-61 double mutant grew on succinate at 60% the efficiency of wild type. Hence the essential carboxyl group of subunit c can function when anchored at either position 24 or position 61, and this supports the idea that these residues may neighbor each other when subunit c is folded in the membrane. The rate of ATP-driven H+ translocation by mutant membrane vesicles was estimated by the quenching of 9-amino-6-chloro-2-methoxyacridine fluorescence and corresponded to actual H+ pumping rates less than 25% that of wild type.
Miller
M J
MJ
Department of Physiological Chemistry, University of Wisconsin Medical School, Madison 53706.
Oldenburg
M
M
Fillingame
R H
RH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D006180:Proton-Translocating ATPases