In 1966, André Jagendorf showed that chloroplasts synthesize ATP in the dark when an artificial pH gradient is imposed across the thylakoid membrane. To create this transient pH gradient, he soaked chloroplasts in a pH 4 buffer for several hours and then rapidly mixed them with a pH 8 buffer containing ADP and P_i. The pH of the stroma suddenly increased to 8, whereas the pH of the thylakoid space remained at 4. A burst of ATP synthesis then accompanied the disappearance of the pH gradient across the thylakoid membrane (Figure 19.24). This incisive experiment was one of the first to unequivocally support the hypothesis put forth by Peter Mitchell that ATP synthesis is driven by proton-motive force (Section 18.4).

The principles of ATP synthesis in chloroplasts are nearly identical with those in mitochondria. ATP formation is driven by a proton-motive force in both photophosphorylation and oxidative phosphorylation. We have seen how light induces electron transfer through photosystems II and I and the cytochrome bf complex. At various stages in this process, protons are released into the thylakoid lumen or taken up from the stroma, generating a proton gradient. The gradient is maintained because the thylakoid membrane is essentially impermeable to protons. The thylakoid space becomes markedly acidic, with the pH approaching 4. The light-induced transmembrane proton gradient is about 3.5 pH units. As discussed in Section 18.4, energy inherent in the proton gradient, called the proton-motive force (Δp), is described as the sum of two components: a charge gradient and a chemical gradient. In chloroplasts, nearly all of Δp arises from the pH gradient, whereas in mitochondria, the contribution from the membrane potential is larger. The reason for this difference is that the thylakoid membrane is quite permeable to Cl⁻ and Mg²⁺. The light-induced transfer of H⁺ into the thylakoid space is accompanied by the transfer of either Cl⁻ in the same direction or Mg²⁺ (1 Mg²⁺ per 2 H⁺) in the opposite direction. Consequently, electrical neutrality is maintained and no membrane potential is generated. The influx of Mg²⁺ into the stroma plays a role in the regulation of the Calvin Cycle (Section 20.2). A pH gradient of 3.5 units across the thylakoid membrane corresponds to a proton-motive force of 0.20 V or a ΔG of −20.0 kJ mol⁻¹ (−4.8 kcal mol⁻¹).

The proton-motive force generated by the light reactions is converted into ATP by the ATP synthase of chloroplasts, also called the CF₁–CF₀ complex (C stands for chloroplast; F for factor). CF₁–CF₀ ATP synthase closely resembles the F₁–F₀ complex of mitochondria (Section 18.4). CF₀ conducts protons across the thylakoid membrane, whereas CF₁ catalyzes the formation of ATP from ADP and P_i.

CF₀ is embedded in the thylakoid membrane. It consists of four different polypeptide chains known as I (17 kDa), II (16.5 kDa), III (8 kDa), and IV (27 kDa) having an estimated stoichiometry of 1 : 2 : 10–14 : 1. Subunits I and II have sequence similarity to subunit b of the mitochondrial F₀ subunit, III corresponds to subunit c of the mitochondrial complex, and subunit IV is similar in sequence to subunit a. CF₁, the site of ATP synthesis, has a subunit composition α₃β₃γΔϵ. The β subunits contain the catalytic sites, similarly to the F₁ subunit of mitochondrial ATP synthase. Remarkably, the β subunits of ATP synthase in corn chloroplasts are more than 60% identical in amino acid sequence with those of human ATP synthase, despite the passage of approximately 1 billion years since the separation of the plant and animal kingdoms.

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Note that the membrane orientation of CF₁–CF₀ is reversed compared with that of the mitochondrial ATP synthase (Figure 19.25). However, the functional orientation of the two synthases is identical: protons flow from the lumen through the enzyme to the stroma or matrix where ATP is synthesized. Because CF₁ is on the stromal surface of the thylakoid membrane, the newly synthesized ATP is released directly into the stromal space. Likewise, NADPH formed by photosystem I is released into the stromal space. Thus, ATP and NADPH, the products of the light reactions of photosynthesis, are appropriately positioned for the subsequent dark reactions, in which CO₂ is converted into carbohydrate.

The activity of the ATP synthase is sensitive to the redox conditions in the chloroplast. For maximal activity, a specific disulfide bond in the γ subunit must be reduced to two cysteines. The reductant is reduced thioredoxin, which is formed from ferredoxin generated in photosystem I, by ferredoxin–thioredoxin reductase, an iron–sulfur containing enzyme.

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Conformational changes in the ϵ subunit also contribute to synthase regulation. The ϵ subunit appears to exist in two conformations. One conformation inhibits ATP hydrolysis by the synthase, while the other, which is generated by an increase in the proton-motive force, allows ATP synthesis and facilitates the reduction of the disulfide bond in the γ subunit. Thus, synthase activity is maximal when biosynthetic reducing power and a proton gradient are available. We will see in Chapter 20 that redox regulation is also important in photosynthetic carbon metabolism.

On occasion, when the ratio of NADPH to NADP⁺ is very high, as might be the case if there was another source of electrons to form NADPH (Section 20.3), NADP⁺ may be unavailable to accept electrons from reduced ferredoxin. In these circumstances, specific large protein complexes allow cyclic electron flow that powers ATP synthesis. Electrons arising from P700, the reaction center of photosystem I, generate reduced ferredoxin. The electron in reduced ferredoxin is transferred to the cytochrome bf complex rather than to NADP⁺. This electron then flows back through the cytochrome bf complex to reduce plastocyanin, which can then be reoxidized by P700⁺ to complete a cycle. The net outcome of this cyclic flow of electrons is the pumping of protons by the cytochrome bf complex. The resulting proton gradient then drives the synthesis of ATP. In this process, called cyclic photophosphorylation, ATP is generated without the concomitant formation of NADPH (Figure 19.26). Photosystem II does not participate in cyclic photophosphorylation, and so O₂ is not formed from H₂O.

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We can now estimate the overall stoichiometry for the light reactions. The absorption of four photons by photosystem II generates one molecule of O₂ and releases four protons into the thylakoid lumen. The two molecules of plastoquinol are oxidized by the Q cycle of the cytochrome bf complex to release eight protons into the lumen. Finally, the electrons from four molecules of reduced plastocyanin are driven to ferredoxin by the absorption of four additional photons. The four molecules of reduced ferredoxin generate two molecules of NADPH. Thus, the overall reaction is

The 12 protons released in the lumen can then flow through ATP synthase. Let us assume that there are 12 subunit III components in CF₀. We expect that 12 protons must pass through CF₀ to complete one full rotation of CF₁. A single rotation generates three molecules of ATP. Given the ratio of 3 ATP for 12 protons, the overall reaction is

Thus, eight photons are required to yield three molecules of ATP (2.7 photons/ATP).

Cyclic photophosphorylation is a somewhat more productive way to synthesize ATP than noncyclic photophosphorylation (the Z scheme). The absorption of four photons by photosystem I leads to the release of eight protons into the lumen by the cytochrome bf system. These protons flow through ATP synthase to yield two molecules of ATP. Thus, each two absorbed photons yield one molecule of ATP. No NADPH is produced.