Abstract
ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ϵ subunits. To clarify the function of the ϵ subunit from phototrophs, here we analyzed the ϵ subunit-mediated inhibition (ϵ-inhibition) of cyanobacterial F1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal α-helix null ϵ-mutants; one lacked the C-terminal α-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two α-helices or an α-helix and a β-sandwich structure. All of these ϵ-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F1-ATPase in the presence of these ϵ-mutants. The stop angular position of the γ subunit in the presence of the ϵ-mutant was identical to that in the presence of the WT ϵ. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80° forcing of the γ subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the ϵ-mutants or by the WT ϵ subunit. These results imply that the C-terminal α-helix domain of the ϵ subunit of cyanobacterial enzyme does not directly inhibit ATP hydrolysis and that its N-terminal domain alone can inhibit the hydrolysis activity. Notably, this property differed from that of the proteobacterial ϵ, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the ϵ subunit-mediated regulation of ATP synthase.
Highlights
ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ⑀ subunits
Because ATP synthase can potentially catalyze ATP hydrolysis when the membrane potential is insufficient for ATP synthesis, regulation of the activity should be important for living cells to avoid futile ATP hydrolysis reactions
The ␥ subunits of chloroplast-type ATP synthases, including the cyanobacterial one, possess an insertion region composed of 30 – 40 amino acids between the Rosmann-fold domain and the C-terminal domain (CTD)2 of ␣-helices, which is not observed in the ␥ subunit of other F1; this region has a function in regulating F1-ATPase [15, 16]
Summary
The ␥ subunits of chloroplast-type ATP synthases, including the cyanobacterial one, possess an insertion region composed of 30 – 40 amino acids between the Rosmann-fold domain and the C-terminal domain (CTD) of ␣-helices, which is not observed in the ␥ subunit of other F1; this region has a function in regulating F1-ATPase [15, 16]. The ␥ subunit of the chloroplast ATP synthase has an additional nine-amino acid insertion containing a pair of Cys residues at this insertion region, and this Cys pair is key for the redox control by thioredoxin [17, 18] Under light conditions, this pair of Cys, which forms a disulfide bond under dark conditions, is reduced by thioredoxin, and the catalytic activity is accelerated [12, 19, 20]. The inhibitory properties of these mutant ⑀ subunits were thoroughly examined
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