Redox Regulation of Rotation of the Cyanobacterial F1-ATPase Containing Thiol Regulation Switch

Abstract

F 1 -ATP synthase (F 1 -ATPase) is equipped with a special mechanism that prevents the wasteful reverse reaction, ATP hydrolysis, when there is insufficient proton motive force to drive ATP synthesis. Chloroplast F 1 -ATPase is subject to redox regulation, whereby ATP hydrolysis activity is regulated by formation and reduction of the disulfide bond located on the ␥ subunit. To understand the molecular mechanism of this redox regulation, we constructed a chimeric F 1 complex (␣ 3 ␤ 3 ␥ redox ) using cyanobacterial F 1 , which mimics the regulatory properties of the chloroplast F 1 -ATPase, allowing the study of its regulation at the single molecule level. The redox state of the ␥ subunit did not affect the ATP binding rate to the catalytic site(s) and the torque for rotation. However, the long pauses caused by ADP inhibition were frequently observed in the oxidized state. In addition, the duration of continuous rotation was relatively shorter in the oxidized ␣ 3 ␤ 3 ␥ redox complex. These findings lead us to conclude that redox regulation of CF 1 -ATPase is achieved by controlling the probability of ADP inhibition via the ␥ subunit inserted region, a sequence feature observed in both cyanobacterial and chloroplast ATPase ␥ subunits, which is important for ADP inhibition (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865).The ATP synthase complex is ubiquitously found in energytransducing membranes such as bacterial plasma membranes, mitochondrial inner membranes, and chloroplast thylakoid membranes, where its basic architecture is highly conserved. ATP synthase consists of two motor units: the F 1 portion which is powered by ATP, and the F o portion powered by the proton motive force formed across the energy transducing membranes (1). Fuelling of ATP synthase by ATP and proton motive force results in rotational motion of F 1 and F o , respectively, though they rotate in opposite directions. On the membranes, these two motors are directly connected by protein-protein interaction, and their functions are coupled to each other: the F 1 portion catalyzes ATP hydrolysis and ATP synthesis, and the F o portion catalyzes proton translocation. Upon formation of a proton motive force across the membranes as a result of respiratory or photosynthetic electron transport, the F o portion is forced to rotate by the proton motive force. Rotation of F o induces rotation of the central axis subunits, ␥ and ⑀, of F 1 , and finally ATP is formed at the catalytic sites on ␤ subunits, presumably due to forced conformational change at the catalytic sites. Vice versa, rotation of F 1 forced by ATP hydrolysis induces rotation of F o and consequently protons are transferred in the opposite direction, where a proton motive force is generated. This enzyme may therefore hydrolyze ATP and transport protons in the opposite direction when there is insufficient proton motive force to drive ATP synthesis.However this reverse reaction catalyzed by the enzyme is highly restricted ...

A Conformational Change of the γ Subunit Indirectly Regulates the Activity of Cyanobacterial F1-ATPase

“…On this point, further analysis is required to understand how the conformational change caused by redox regulation is transferred to the catalytic site(s) in CF 1 enzyme molecule. Previously, we constructed a chimeric cyanobacterial F 1 complex in which the 9-amino acid region of spinach CF 1 -␥ including two regulatory Cys was introduced into the cyanobacterial ␥ subunit (36). Based on the single molecular observation of rotation of this chimeric complex, we concluded that redox regulation is achieved by controlling the probability to lapse into ADP inhibition.…”

“…Although the major mechanism that acts to regulate its activity is ADP inhibition of the catalytic sites (11,12,14,42), the reason that sites of redox regulation and the ⑀ inhibition are structurally distant from the catalytic site is poorly understood. We postulated that the relative slippage of the two central ␣-helices of the ␥ subunit, which connect the two active domains for catalysis and regulation in the structure of the complex (8), must be a main cause of the change in the enzyme activity (36). To examine this hypothesis, we prepared several mutant complexes in which the relative slippage of two central ␣-helices can be locked at a certain position by disulfide bond formation.…”

“…Since little contact region is reported in the crystal structure of F 1 between the ␥ subunit and the ␣ 3 ␤ 3 ring (Fig. 1A), we postulated that relative slippage of N-and C-terminal ␣-helices may connect between the conformational change of the bottom part of ␥ and a conformational change in the upper part of ␥, as suggested by our previous study on the redox regulation of this enzyme complex (36).…”

Regulation of F0F1-ATPase from Synechocystis sp. PCC 6803 by γ and ∈ Subunits Is Significant for Light/Dark Adaptation

“…This might be related to the fact that both ␥ and ⑀ subunits play critical roles in regulation of the activity of the enzyme (5,6). In comparison with bacterial or mitochondrial ATPases, the chloroplast F 1 (CF 1 ) ␥ subunit contains an additional inserted region of ϳ35 residues, within which two cysteine residues allow redox regulation of ATPase activity by disulfide bond formation/elimination (7)(8)(9)(10). Notably, though containing a similar insertion, the ␥ subunit of cyanobacterial F 1 lacks nine amino acids therein, including the two regulatory cysteines (11).…”

Redox regulation of CF1-ATPase involves interplay between the γ-subunit neck region and the turn region of the βDELSEED-loop

“…Cyanobacteria do not possess regulatory cysteines in their γ-subunits, although a similar γ-insertion can be found [20,25]. However, the regulation mechanism found in CF 1 was successfully transferred to a cyanobacterial enzyme by insertion of the cysteine-containing fragment from spinach CF 1 into the globular domain of the prokaryotic γ-subunit [26]. A major benefit of studying the ATPase redox regulation is the preservation of crucial interactions between the γ-stalk and the α 3 β 3 hexamer, despite the insertion.…”