Abstract
The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF1) and E. coli F1F0 ATP synthase (ECF1F0) have been isolated from a novel mutant gammaY205C. ECF1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine. This effect was not seen in ECF1F0. The mutation partly disrupts the F1 to F0 interaction, as indicated by a reduced efficiency of proton pumping. ECF1 containing the mutation gammaY205C was bound to the membrane-bound portion of the E. coli F1F0 ATP synthase (ECF0) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes. Cu2+ treatment or reaction with 5,5'-dithio-bis(2-nitro-benzoic acid) induced disulfide bond formation between the Cys at gamma position 205 and a Cys residue at positions 42, 43, or 44 in the c subunit but not at position 39. Using Cu2+ treatment, this covalent cross-linking was obtained in yields as high as 95% in the hybrid ECF1 gammaY205C/cQ42C and in ECF1F0 isolated from the double mutant of the same composition. The covalent linkage of the gamma to a c subunit had little effect on ATPase activity. However, ATP hydrolysis-linked proton translocation was lost, by modification of both gamma Cys-205 and c Cys-42 by bulky reagents such as 5,5'-dithio-bis (2-nitro-benzoic acid) or benzophenone-4-maleimide. In both ECF1 and ECF1F0 containing a Cys at gamma 205 and a Cys in the epsilon subunit (at position 38 or 43), cross-linking of the gamma to the epsilon subunit was induced in high yield by Cu2+. No cross-linking was observed in hybrid enzymes in which the Cys was at position 10, 65, or 108 of the epsilon subunit. Cross-linking of gamma to epsilon had only a minimal effect on ATP hydrolysis. The reactivity of the Cys at gamma 205 showed a nucleotide dependence of reactivity to maleimides in both ECF1 and ECF1F0, which was lost in ECF1 when the epsilon subunit was removed. Our results show that there is close interaction of the gamma and epsilon subunits for the full-length of the stalk region in ECF1F0. We argue that this interaction controls the coupling between nucleotide binding sites and the proton channel in ECF1F0.
Highlights
Richia coli enzyme is composed of three different subunits: a, b, and c in the molar ratio 1:2:10 –12 [1,2,3,4]
Characterization of Escherichia coli F1F0 ATP synthase (ECF1) and E. coli F1F0 ATP synthase (ECF1F0) Isolated from the Mutant ␥Y205C—ECF1 isolated from the mutant ␥Y205C contained ␣, , ␥, ␦, and ⑀ subunits in the same relative amounts as wild-type strain, whereas ECF1F0 isolated from the mutant had the same subunit composition as wild type based on SDS
␥Y205C, had an increased ATPase activity due to an altered affinity of the ␣33 ␥ core complex for the inhibitory ⑀ subunit, but the ATPase activity of the intact ECF1F0 was not altered compared with wild type by the mutation
Summary
Richia coli enzyme is composed of three different subunits: a, b, and c in the molar ratio 1:2:10 –12 [1,2,3,4]. The recently published high resolution structure of a major part of the beef heart F1 molecule confirms the above-described arrangement of the ␣, , and ␥ subunits and adds important details [11] It shows the ␥ subunit arranged with a long C-terminal ␣-helix extending from the top of the ␣ and  subunits into the stalk region. We observed cross-linking between a Cys introduced at position 44 of the polar loop of the c subunits and a site or sites on the ␥ subunit in the region between residues 202 and 230 [12] This result implies that the ␥ subunit extends the full length of the stalk to interact with the F0 part in the intact ATP synthase. This is the same region of ␥ that was identified in preliminary experiments as involved in binding to the c subunits [12]
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