THE GAMMA-SUBUNIT OF THE ESCHERICHIA-COLI F(1)-ATPASE CAN BE CROSS-LINKED NEAR THE GLYCINE-RICH LOOP REGION OF A BETA-SUBUNIT WHEN ADP+MG(2+) OCCUPIES CATALYTIC SITES BUT NOT WHEN ATP+MG(2+) IS BOUND

A mutant of the Escherichia coli F1-ATPase, gammaS8C, has been reacted with a novel bifunctional reagent, aleimido-N'-(4-azido-2,3,5,6-tetra fluorobenzamido) cystamine (TFPAM-SS1). Modification of Cys-8 via the maleimide, followed by photolysis to convert the azido group to a reac tive nitrene, led to cross-linking of the gamma subunit to a beta subu nit. When this cross-linking was conducted with ADP + Mg2+ in catalyti c sites, the predominant cross-linked product had a M(r) of 108,000. I f cross-linking was done with uncleaved ATP + Mg2+ in catalytic sites, cross-linked products of 102,000 and 84,000 were formed. Cross-linkin g under both conditions led to inhibition of ATPase activity. TFPAM-SS 1 could be cleaved by using reducing agents to break the disulfide bon d that links the maleimide and tetrafluorophenylazide moieties. Cleava ge of this disulfide bond after formation of 102,000 and 84,000 specie s led to full recovery of ATPase activity. When the 108-kDa cross-link ed product was cleaved, full activity was not restored, presumably bec ause of insertion of the tetrafluorophenylazide into a functionally im portant site on the beta subunit. After cleavage of the disulfide bond , the free thiols could be reacted with [C-14]N-ethylmaleimide, thereb y radioactively tagging the sites of insertion of the tetrafluoropheny lnitrene moiety. In this way, the site of cross-linking from Cys-8 of gamma to the beta subunit in the presence of ADP + Mg2+ was localized to within the sequence Val 145-Lys-155, which contains the glycine-ric h loop. This loop region is a part of the catalytic site of the enzyme .