The defective proton-ATPase of uncA mutants of Escherichia coli. Studies of nucleotide binding sites, bound aurovertin fluorescence, and labeling of essential residues of the purified F1-ATPase.

Abstract

The Escherichia coli uncA gene codes for the a subunit of the F1-sector of the membrane proton-ATPase.Mutations in this gene cause loss of ATPase and ATP synthesis activity and, in some instances, derangement of F1 structure.From three mutants (uncA401, uncA453, and uncA447), an F1 of normal size and subunit structure may be purified.In this work, purified soluble F1 from these three mutants has been compared with F1 from a normal strain in respect to (a) binding of ATP and ADP, both in the presence and absence of Mg, ( b ) effects of aurovertin on the binding of ATP and ADP, (c) binding of aurovertin itself, ( d ) effects of ADP on the fluorescence of bound aurovertin, (e) reactivity of essential residues using dicyclohexylcarbodiimide and 4-chloro-7-nitrobenzofurazan to modify carboxyl and tyrosyl residues, respectively.UncA F1 was normal or similar to normal in ATP-and ADP-binding capability.An increase in binding affinity for ADP and ATP was induced in normal F1 by aurovertin, but was not induced in uncA F1.Both normal F1 and uncA F1 bound -2 mol of aurovertin/mol of Fl with Kd of approximately 3 p ~, the only difference being that the bound aurovertin showed 70% higher fluorescence in uncA F1 as opposed to normal F1.Whereas ADP induced marked enhancement of bound aurovertin fluorescence in normal F1 (Km ADP -1 to 2 p ~) , no such effect of ADP was seen in uncA F1.The reactivity of essential carboxyl and tyrosyl residues was similar in uncA and normal FI.It is suggested that a t , inter-subunit conformational interaction is abolished in F1 from these three uncA mutants and it is hypothesized that such interaction is required for normal F1-catalyzed ATP hydrolysis and synthesis.