Location of Conserved Residue Histidine-38 of the ϵ-Subunit of Escherichia coli ATP Synthase

The function and location of residue His-38 of the ϵ subunit of the Escherichia coli F1-ATPase were investigated. His-38 was replaced by glutamine and cysteine through site-directed mutagenesis to produce ϵH38Q and ϵH38C, respectively. Both ϵH38Q and ϵH38C fulfilled ϵ function in vivo as determined by growth on nonfermentable carbon sources, growth yield on limiting glucose, and recovery of cells from energy starvation conditions. ϵH38Q and ϵH38C were purified and studied in vitro. Pure ϵH38C reacted rapidly with Ellman′s reagent, indicating a surface location of the introduced cysteine. ϵH38C which had been reconstituted with ϵ-depleted F1-ATPase could be linked specifically to the γ subunit using two different heterobifunctional sulfhydril-reactive/ photoreactive crosslinking agents, indicating that residue 38 lies near γ. The mutated ϵ subunits were unaltered in their ability to inhibit ϵ-depleted F1-ATPase in vitro, even after modification of ϵH38C with the bulky reagents fluorescein maleimide and N-(1-anilinonaphthyl-4)maleimide. It seems unlikely, therefore, that residue His-38 of ϵ interacts directly with y. Both the ϵH38Q and ϵH38C mutations reduced the recognition of by monoclonal antibody ϵ-1, but recognition of ϵH38C was not further reduced by reaction with fluorescein maleimide. These results imply that residue 38 is not directly part of the ϵ-1 epitope, but plays a role in its formation.