Structure, mechanism, and regulation of the chloroplast ATP synthase

Abstract

INTRODUCTION Green plant chloroplasts convert light into chemical energy, and adenosine triphosphate (ATP) generated by photosynthesis is the prime source of biologically useful energy on the planet. Plants produce ATP by the chloroplast F 1 F o ATP synthase (cF 1 F o ), a macromolecular machine par excellence, driven by the electrochemical proton gradient across the photosynthetic membrane. It consists of 26 protein subunits, 17 of them wholly or partly membrane-embedded. ATP synthesis in the hydrophilic α 3 β 3 head (cF 1 ) is powered by the cF o rotary motor in the membrane. cF o contains a rotor ring of 14 c subunits, each with a conserved protonatable glutamate. Subunit a conducts the protons to and from the c-ring protonation sites. The central stalk of subunits γ and e transmits the torque from the F o motor to the catalytic cF 1 head, resulting in the synthesis of three ATP per revolution. The peripheral stalk subunits b, b′, and δ act as a stator to prevent unproductive rotation of cF 1 with cF o . All rotary ATP synthases are, in principle, fully reversible. To prevent wasteful ATP hydrolysis, cF 1 F o has a redox switch that inhibits adenosine triphosphatase (ATPase) activity in the dark. RATIONALE Understanding the molecular mechanisms of this elaborate nanomachine requires detailed structures of the whole complex, ideally at atomic resolution. Because of the dynamic nature of this membrane protein complex, crystallization has been difficult and no high-resolution structure of an entire, functional ATP synthase is available. We reconstituted cF 1 F o from spinach chloroplasts into lipid nanodiscs and determined its structure by cryo–electron microscopy (cryo-EM). Cryo-EM is the ideal technique for this study because it can deliver high-resolution structures of large, dynamic macromolecular assemblies that adopt a mixture of conformational states. RESULTS We present the cryo-EM structure of the intact cF 1 F o ATP synthase in lipid nanodiscs at a resolution of 2.9 A (cF 1 ) to 3.4 A (cF o ). In the cF 1 ATPase head, we observe nucleotides with their coordinating Mg ions and water molecules, allowing assignment to the three well-characterized functional states involved in rotary ATP synthesis. Subunit δ on top of the ATPase head binds to all three α subunits, ensuring that only one peripheral stalk can attach. The loosely entwined, long α helices of the peripheral stalk subunits b and b′ clamp the integral membrane subunit a in its position next to the c-ring rotor, thus connecting cF 1 to cF o . Subunit γ has an L-shaped double hairpin with a redox sensor that can form a disulfide bond and a chock that blocks rotation to avoid wasteful ATP hydrolysis at night. Protons are translocated through access routes in subunit a in all rotary ATPases. We observe a hydrophilic channel on the lumenal surface that connects to the glutamate residues on the c-ring rotor that carry protons for an almost full rotation before releasing them into the stroma through another hydrophilic channel. A strictly conserved arginine separates the access and exit channels, preventing leakage of protons through the membrane. CONCLUSION We observe three cF 1 F o conformations, each with the central rotor stalled in a different position. Ring rotation is unexpectedly divided into three unequal steps. The peripheral stalk may thus act like an elastic spring, evening out the different energy contributions of each step. The features of ATP synthase nanomachines are remarkably similar in chloroplasts and mitochondria, considering their evolutionary distance of a billion years or more.