ASYMMETRY OF ESCHERICHIA-COLI F1-ATPASE AS A FUNCTION OF THE INTERACTION OF ALPHA-BETA SUBUNIT PAIRS WITH THE GAMMA-SUBUNIT AND EPSILON-SUBUNIT

The asymmetry of Escherichia coli F-1-ATPase (ECF(1)) has been explored in chemical modification experiments involving two mutant enzyme preparations. One mutant contains a cysteine (Cys) at position 149 of the beta subunit, along with conversion of a Val to Ala at residue 198 to suppress the deleterious effect of the Cys for Gly at 149 mutation (mutant beta G149C:V198A). The second mutant has these mutations and also Cys residues at positions 381 of beta and 108 of the epsilon subunit (mutant beta G149C:V198A:E381C/epsilon S108C). On CuCl2 treatment of this second mutant, there is cross-linking of one copy of the beta subunit to gamma via the Cys at 381, a second to the epsilon subunit (between beta Cys(381) and epsilon Cys(108)), while the third beta subunit in the ECF, complex is mostly free (some crosslinking to delta); thereby distinguishing the three beta subunits as beta(gamma), beta(epsilon), and beta(free), respectively. Both mutants have ATPase activities similar to wild-type enzyme. Under all nucleotide conditions, including with essentially nucleotide-free enzyme, the three different beta subunits were found to react differently with N-ethylmaleimide (NEM) which reacts with Cys(149) dicyclohexyl carbodiimide (DCCD) which reacts with Glu(192), and 7-chloro-4-nitrobenzofurazan (NbfCl) which reacts with Tyr(297). Thus, beta(gamma) reacted with BOOB but not NEM or NbfCl; beta(free) was reactive with all three reagents; beta(epsilon), reacted with NEM, but was poorly reactive to DCCD or NbfCl. There was a strong nucleotide dependence of the reaction of Cys(149) in beta(epsilon) (but not in beta(free)) with NEM, indicative of the important role that the epsilon subunit plays in functioning of the enzyme.