Modulation of Nucleotide Specificity of Thermophilic FoF1-ATP Synthase by ϵ-Subunit

The C-terminal two α-helices of the ϵ-subunit of thermophilic Bacillus FoF1-ATP synthase (TFoF1) adopt two conformations: an extended long arm (“up-state”) and a retracted hairpin (“down-state”). As ATP becomes poor, ϵ changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TFoF1 expressed in Escherichia coli, we compared TFoF1 with up- and down-state ϵ in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TFoF1 with the up-state ϵ was achieved by inclusion of hexokinase in the assay and TFoF1 with the down-state ϵ was represented by ϵΔc-TFoF1, in which ϵ lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TFoF1 with the down-state ϵ synthesizes GTP at the same rate of ATP, whereas TFoF1 with the up-state ϵ synthesizes GTP at a half-rate. Though rates are slow, TFoF1 with the down-state ϵ even catalyzes UTP and CTP synthesis. Authentic TFoF1 from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the down-state. NTP hydrolysis and NTP-driven proton pumping activity of ϵΔc-TFoF1 suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ϵ-subunit modulates substrate specificity of TFoF1.