SUBUNITS COUPLING H-COLI ATP SYNTHASE - CYS-CYS CROSS-LINKLNG OF F1 SUBUNIT-EPSILON TO THE POLAR LOOP OF F-0 SUBUNIT-C( TRANSPORT AND ATP SYNTHESIS IN THE ESCHERICHIA)

Second site suppressor mutations at position 31 of F-1 subunit epsilon recouple ATP-driven H+ translocation in the uncoupled Q42E mutant of subunit c of the Escherichia coli F1F0 ATP synthase (Zhang, Y,, Oldenb urg, M,, and Fillingame, R, H, (1994) J, Biol. Chem, 269, 10221-10224) , This finding suggests a functional interaction between subunit c and subunit epsilon during the coupling of H+ transport through F-0 to AT P synthesis of F-1, However, the physical proximity of the two subunit s remained to be defined, In this study, Cys residues were introduced into residues in the polar loop region of subunit c surrounding Gln42 and at position 31 of subunit epsilon to see whether the subunits coul d be cross-linked, Disulfide bridge formation between subunit c and su bunit epsilon was observed in membranes of three double mutants, i.e. cA40C/epsilon E31C, cQ42C/epsilon E31C, and cP43C/epsilon E31C, but no t in wild type membranes or in membranes of the cA39C/epsilon 31C doub le mutant, These results indicate that the polar loop of subunit c and the region around residue 31 of subunit epsilon are physically close to each other in the F1F0 complex and support the hypothesis that thes e two subunits interact directly in the coupling of H+ transport to AT P synthesis, Disulfide cross-linking of the Q42C subunit c and E31C su bunit epsilon leads to inhibition of ATPase coupled H+ transport, as m ight be expected in a model where the catalytic sites of the F-1 ATPas e alternate during H+ transport-coupled ATP hydrolysis/synthesis. Howe ver, a quantitative relationship between the extent of inhibition of t ransport and the extent of crosslinking could not be established by th e methods used here, and the possibility remains that the epsilon-C cr oss-linked F1F0 complex retains residual H+ transporting activity.