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The Essential Carboxyl Group in Subunit c of the F1F0 ATP Synthase can be Moved and H+-Translocating Function Retained

Michael J. Miller, Mary Oldenburg and Robert H. Fillingame
Proceedings of the National Academy of Sciences of the United States of America
Vol. 87, No. 13 (Jul., 1990), pp. 4900-4904
Stable URL: http://www.jstor.org/stable/2354519
Page Count: 5
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
The Essential Carboxyl Group in Subunit c of the F1F0 ATP Synthase can be Moved and H+-Translocating Function Retained
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Abstract

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 < 25% that of wild type.

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