The N-terminal region of the ε subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity

Abstract

Edited by Joseph M. Jez ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ⑀ subunits. To clarify the function of the ⑀ subunit from phototrophs, here we analyzed the ⑀ subunit-mediated inhibition (⑀-inhibition) of cyanobacterial F 1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal ␣-helix null ⑀-mutants; one lacked the C-terminal ␣-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two ␣-helices or an ␣-helix and a ␤-sandwich structure. All of these ⑀-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F 1-ATPase in the presence of these ⑀-mutants. The stop angular position of the ␥ subunit in the presence of the ⑀-mutant was identical to that in the presence of the WT ⑀. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80°forcing of the ␥ subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the ⑀-mutants or by the WT ⑀ subunit. These results imply that the C-terminal ␣-helix domain of the ⑀ 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 ⑀, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the ⑀ subunit-mediated regulation of ATP synthase.

Structure, Regulation, and Significance of Cyanobacterial and Chloroplast Adenosine Triphosphate Synthase in the Adaptability of Oxygenic Photosynthetic Organisms

“…The ε subunit of cyanobacteria also plays a vital role in regulating the activity of ATP synthase. Unlike in E. coli, the CTD of the ε subunit of cyanobacterial ATP synthase alone does not inhibit the degradation activity of ATP synthase [71]. The CTD is not the functional domain of the ε subunit in regulating the activity of cyanobacterial ATP synthase.…”

“…Additionally, the phosphorylation of two tryptophan residues at the NTD of the β subunit is important for the accumulation of chloroplast ATP synthase, and is potentially involved in the assembly of ATP synthase [65]. Besides Atp1, which participates in the assembly of the F o -c-ring [11], no accessory ATP synthase assembly proteins have been reported in cyanobacteria, while functional cyanobacterial F 1 complexes have successfully been expressed in E. coli by transforming the F 1 -encoding genes into the bacterium [66][67][68][69][70][71], suggesting that the assembly process of the F 1 complex in cyanobacteria may be similar to that in E. coli and the accessory proteins involved in both systems may possess functional similarities.…”

“…Instead, the NTD directly participates in the inhibition process. Independent of the CTD, the NTD itself possesses an inhibitory effect on ATP hydrolysis [71]. The crystal structure of the γε complex suggests a tight protein-protein interaction between ε-NTD and the γ subunit [26], which is involved in ADP-induced inhibition during ATP hydrolysis [66], implying that ε-NTD and the γ subunit possibly cooperate to inhibit the degradation activity of cyanobacterial ATP synthase [26,89].…”

The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria

“…This was possibly explained by the interaction between the DELSEED region of the β subunit and a part of the γ subunit, although structural studies are necessary to prove this hypothesis. It was recently reported that the ε subunit of cyanobacterial F o F 1 has a different inhibitory mechanism compared with other organisms ( 41 ) and that the binding of the ε subunit caused a relative conformational change in the γ subunit ( 32 ). Consequently, the β-hairpin structure of the S. 6803 γ subunit, assisted by the ε subunit, might confer stiffness to the γ subunit and facilitate torque transmission during ATP synthesis.…”