The epsilon subunit of chloroplast ATP synthase: Effects of C-terminal deletions on enzyme activity and regulation

The chloroplast ATP synthase is the enzyme that utilizes the electrochemical proton gradient established during photosynthesis for the production of ATP. The enzyme, located on the thylakoid membrane, has two major domains, the extrinsic CF₁ and the membrane intrinsic CF_o, each composed of several subunits. This very complex enzyme also possesses complex regulation to prevent ATP hydrolysis in the dark. One of the modes of regulation is the small ϵ subunit of CF₁. This subunit has two domains, an N-terminal β-sandwich and C-terminal helix-loop-helix. The ϵ subunit is not required for the binding of the catalytic portion of CF₁ to CF_o. It has been shown that the ϵ subunit is required for inhibition of ATP hydrolysis, but is also required for ATP synthesis. Thylakoid membranes containing CF₁ lacking ϵ (CF₁-ϵ) have high ATPase activity and no ATP synthesis activity. When either recombinant or native ϵ is added to these membranes, ATP synthesis is restored and ATPase activity is inhibited. Recombinant ϵ subunit lacking the last 45 amino acids, corresponding to the helix-loop-helix, was able to restore ATP synthesis to thylakoid membranes containing CF₁-ϵ, but was unable to prevent ATP hydrolysis by these membranes. In addition, these membranes exhibit lower levels of proton gradient formation in the light, and high background levels of ATP hydrolysis in the presence of an artificial proton gradient. Thus, the C-terminus of ϵ is involved in regulation, by inhibiting the ATP synthase, but is not required for ATP synthesis.