CLOSE PROXIMITY OF CYS(64) AND CYS(140) IN THE DELTA-SUBUNIT OF ESCHERICHIA-COLI F1-ATPASE

The delta subunit of the F-1-ATPase from Escherichia coli contains 2 c ysteine residues, one at position 64 and the second at position 140 of the amino acid sequence. These residues were specifically labeled wit h sulfhydryl reagents in this study without labeling other -SH groups in the enzyme. Modification of Cys(140) by maleimides such as N-ethylm aleimide or fluorescein maleimide resulted in a reconstitutively activ e enzyme that was indistinguishable from the native protein. Labeling of Cys(64) with Or without concomitant labeling of Cys(140) resulted i n a reconstitutively inactive enzyme. The ATPase activity of either fo rm of the labeled enzyme was unaffected. However, labeling of Cys(64) was accompanied by dissociation of the delta subunit from the enzyme. These observations suggest a role for the microenvironment of Cys(64) in interactions of the delta subunit with other subunits in the enzyme . Two types of evidence support the conclusion that the 2 cysteine res idues of the delta subunit are in close proximity. First, incorporatio n of pyrene maleimide into both delta cysteines led to excimer formati on. Second, incubation of F-1 with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in quantitative formation of a disulfide bond between Cys(64 ) and Cys(140), presumably via disulfide interchange. The enzyme conta ining the internally cross-linked delta subunit exhibited an undiminis hed ability to support proton pumping when reconstituted into F-1-depl eted membrane vesicles. The presence of 2 closely apposed cysteinyl re sidues in the delta subunit of the native enzyme places constraints on the type of structure that may be proposed for the subunit.