Abstract
The substitution of arginine at position 210 in the alpha subunit of Escherichia coli F0F1-ATPase by either lysine or alanine causes dominance in complementation tests with a chromosomal c subunit mutation. Reversal of dominance was achieved for the alpha R210K mutation but not for the alpha R210A mutation by the presence of an aspartic acid residue at position 50 or at position 252 in the alpha subunit. It was concluded that position 210 in putative helix 4 of a previously proposed model of the alpha subunit is close to position 252 in putative helix 5 and to position 50 in putative helix 1. The juxtaposition of residues 252 and 210 was also indicated by the observation that the double mutant alpha R210Q/Q252R was partially functional. A revertant of the partially functional double mutant, isolated on succinate medium, was found to contain a third mutation resulting in Pro-204 in the alpha subunit being replaced by threonine. That the revertant phenotype was due to the alpha P204T change was confirmed by site-directed mutagenesis. ATP synthesis in the revertant strain was at near normal levels as judged by growth yield experiments, but the revertant strain was unable to pump protons in response to ATP hydrolysis.
Highlights
The F0F1-ATPase enzyme complex catalyses the terminal step in oxidative phosphorylation and photophosphorylation and is located in mitochondrial, chloroplast, and bacterial membranes
The a, b, and c subunits of the proton pore are present in a stoichiometry of 1:2:6 –12 [2], and all are required for proton translocation
The remaining two residues required for proton translocation are not strictly conserved, and some amino acid substitutions at either position retain some proton translocating activity (4 – 6, 10, 11)
Summary
Enzymes and Chemicals—All chemicals and enzymes used were of the highest quality commercially available. The mineral salts minimal medium used and additions were as described previously [21]. The minimal salts medium in the fermenters was supplemented with 5% (v/v) Luria broth [23]. Growth yields were measured as turbidities after growth had ceased in minimal medium containing limiting (5 mM) glucose and supplemented with 5% Luria broth as well as specific requirements. Preparation of Plasmid and Phage M13 DNA—Single-stranded “template” DNA was prepared as described for the oligonucleotide-directed in vitro mutagenesis system (Amersham Corp.). Replicative form DNA and plasmid DNA were prepared by the alkaline lysis method [24].
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