The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system.

The F(o)F(1)-ATPase, which synthesizes ATP with a rotary motion, is highly regulated in vivo in order to function efficiently, although there remains a limited understanding of the physiological significance of this regulation. Compared with its bacterial and mitochondrial counterparts, the gamma subunit of cyanobacterial F(1), which makes up the central shaft of the motor enzyme, contains an additional inserted region. Although deletion of this region results in the acceleration of the rate of ATP hydrolysis, the functional significance of the region has not yet been determined. By analysis of rotation, we successfully determined that this region confers the ability to shift frequently into an ADP inhibition state; this is a highly conserved regulatory mechanism which prevents ATP synthase from carrying out the reverse reaction. We believe that the physiological significance of this increased likelihood of shifting into the ADP inhibition state allows the intracellular ATP levels to be maintained, which is especially critical for photosynthetic organisms.

The ␥ and ⑀ subunits of F 0 F 1 -ATP synthase from photosynthetic organisms display unique properties not found in other organisms. Although the ␥ subunit of both chloroplast and cyanobacterial F 0 F 1 contains an extra amino acid segment whose deletion results in a high ATP hydrolysis activity (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865), its ⑀ subunit strongly inhibits ATP hydrolysis activity. To understand the physiological significance of these phenomena, we studied mutant strains with (i) a C-terminally truncated ⑀ (⑀ ⌬C ), (ii) ␥ lacking the inserted sequence (␥ ⌬198 -222 ), and (iii) a double mutation of (i) and (ii) in Synechocystis sp. PCC 6803. Although thylakoid membranes from the ⑀ ⌬C strain showed higher ATP hydrolysis and lower ATP synthesis activities than those of the wild type, no significant difference was observed in growth rate and in intracellular ATP level both under light conditions and during light-dark cycles. However, both the ⑀ ⌬C and ␥ ⌬198 -222 and the double mutant strains showed a lower intracellular ATP level and lower cell viability under prolonged dark incubation compared with the wild type. These data suggest that internal inhibition of ATP hydrolysis activity is very important for cyanobacteria that are exposed to prolonged dark adaptation and, in general, for the survival of photosynthetic organisms in an ever-changing environment.

F-ATPase forms the membrane-associated segment of FF-ATP synthase - the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F subcomplex, consisting of the rotary shaft γ subunit and the inhibitory ε subunit, from the photosynthetic cyanobacterium BP-1, at 1.98 Å resolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35-40 amino acids. Our structural data reveal that this region forms a β-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this β-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F subcomplex. Biochemical analyses of the corresponding αβγ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the αβ complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the β-hairpin insertion structure resulted in a marked impairment of the interaction with the ε subunit, which binds to the opposite side of the γ subunit from the β-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F rotation during ATP hydrolysis.

Background: A conformational change of the ␥ subunit of ATP synthase may be critical for enzyme regulation. Results: A conformational change of ␥ controls both ADP inhibition and ⑀ inhibition. Conclusion: The ␥ subunit indirectly regulates the activity by way of ADP inhibition and ⑀ inhibition. Significance: Regulation system of ATP synthase based on the unique molecular structure of the ␥ subunit was revealed.