LOCATION OF CONSERVED RESIDUE HISTIDINE-38 OF THE EPSILON-SUBUNIT OF ESCHERICHIA-COLI ATP SYNTHASE

The function and location of residue His-38 of the ε{lunate} subunit of the Escherichia coli F1-ATPase were investigated. His-38 was replaced by glutamine and cysteine through site-directed mutagenesis to produce ε{lunate}H38Q and ε{lunate}H38C, respectively. Both ε{lunate}H38Q and ε{lunate}H38C fulfilled ε{lunate} function in vivo as determined by growth on nonfermentable carbon sources, growth yield on limiting glucose, and recovery of cells from energy starvation conditions. ε{lunate}H38Q and ε{lunate}H38C were purified and studied in vitro. Pure ε{lunate}H38C reacted rapidly with Ellman′s reagent, indicating a surface location of the introduced cysteine. ε{lunate}H38C which had been reconstituted with ε{lunate}-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{lunate} subunits were unaltered in their ability to inhibit ε{lunate}-depleted F1-ATPase in vitro, even after modification of ε{lunate}H38C with the bulky reagents fluorescein maleimide and N-(1-anilinonaphthyl-4)maleimide. It seems unlikely, therefore, that residue His-38 of ε{lunate} interacts directly with y. Both the ε{lunate}H38Q and ε{lunate}H38C mutations reduced the recognition of by monoclonal antibody ε{lunate}-1, but recognition of ε{lunate}H38C was not further reduced by reaction with fluorescein maleimide. These results imply that residue 38 is not directly part of the ε{lunate}-1 epitope, but plays a role in its formation. © 1993 Academic Press. All rights reserved.