Purification and Reconstitution of the FIFO-ATP Synthase from Alkaliphilic Bacillus firmus OF4 EVIDENCE THAT THE ENZYME TRANSLOCATES

The FIFO-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, in good yield and with a high specific ATPase activity when appropriately activated. The purification procedure involved octyl glucoside extraction of washed membrane vesicles in the presence of 20% glycerol and asolectin followed by ammonium sulfate fractionation and sucrose density gradient centrifugation. The purified preparation was resolved into seven bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to the five F1 subunits, a, B, y, 6, and t, and to the b and c subunits of the Fo. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel analysis revealed a candidate for the a subunit of Fo. The MgATPase activity of B. firmus OF4 FIFo was barely detectable but could be stimulated, optimally more than loo-fold, by sulfite, methanol, and octyl thioglucoside. The enzyme was inhibited by N,N’-dicyclohexylcarbodiimide and sodium azide, but not by aurovertin, an inhibitor of the F1 from Escherichia coli. The FIFo reconstituted into proteoliposomes catalyzed ATPase activity, ATP-Pi exchange, and ATP-dependent ApH and A+ formation. ATP hydrolysis was stimulated by protonophores while the other activities were abolished by protonophores. These activities were neither dependent on added sodium ions nor significantly affected by them. FIFO proteoliposomes made from crude octyl glucoside extracts that also contained the Na+/H+ antiporter were shown to catalyze ATP-dependent Na+ uptake that was completely sensitive to carbonyl cyanide m-chlorophenylhydrazone; Na+ uptake activity was absent in proteoliposomes containing more purified FIFo but lacking the Na+/H+ antiporter. These data show that the FIFO translocates protons and does not substitute Na+ for H’ in energy coupling.