Mutations in single hairpin units of genetically fused subunit c provide support for a rotary catalytic mechanism in F0F1 ATP synthase

Previously, we generated genetically fused dimers and trimers of subunit c of the Escherichia coli ATP synthase based upon the precedent of naturally occurring dimers in V-type H+-transporting ATPases, The c(2) and c, oligome rs have proven useful in testing hypothesis regarding the mechanism of ener gy coupling. In the first part of this paper, the uncoupling Q42E substitut ion has been introduced into the second loop of the c(2) dimer or the third loop of the c(3) trimer, Both mutant proteins proved to be as functional a s the wild type c(2) dimer or wild type c(3) trimer, The results argue agai nst an obligatory movement of the epsilon subunit between loops of monomeri c subunit c in the c(12), oligomer during rotary catalysis, Rather, the res ults support the hypothesis that the c-epsilon connection remains fixed as the c-oligomer rotates. In the second section of this paper, we report on t he effect of substitution of the proton translocating Asp(61) in every seco nd helical hairpin of the c(2) dimer, or in every third hairpin of the c(3) trimer, Based upon the precedent of V-type ATPases, where the c(2) dimer o ccurs naturally with a single proton translocating carboxyl in every second hairpin, these modified versions of the E, coli c(2) and c(3) fused protei ns were predicted to have a functional H+-transporting ATPase activity, wit h a reduced H+/ATP stoichiometry, but to be inactive as ATP synthases, A va riety of Asp(61)-substituted proteins proved to lack either activity indica ting that the switch in function in V-type ATPases is a consequence of more than a single substitution.