The ε Subunit of the F1F0 Complex ofEscherichia coli

Four double mutants in the ε subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds. Two of these were designed to generate interdomain cross-links that lock the C-terminal α-helical domain against the β-sandwich (εM49C/A126C and εF61C/V130C). The second set should give cross-linking between the two C-terminal α-helices (εA94C/L128C and εA101C/L121C). All four mutants cross-linked with 90–100% efficiency upon CuCl2 treatment in isolated Escherichia coli ATP synthase. This shows that the structure obtained for isolated ε is essentially the same as in the assembled complex. Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal α-helices. None of the cross-links had any effect on proton pumping-coupled ATP hydrolysis, on DCCD sensitivity of this activity, or on ATP synthesis rates. Therefore, big conformational changes within ε can be ruled out as a part of the enzyme function. Protease digestion studies, however, showed that subtle changes do occur, since the ε subunit could be locked in an ADP or 5′-adenylyl-β,γ-imidodiphosphate conformation by the cross-linking with resulting differences in cleavage rates.

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