A Proton Gradient Across the Thylakoid Membrane Drives ATP Synthesis

II. Transducing and Storing Energy
19. The Light Reactions of Photosynthesis
19.3. Two Photosystems Generate a Proton Gradient and NADPH in Oxygenic Photosynthesis
Figure 19.23. Structure and Function of Ferredoxin-NADP+ Reductase. (A) Structure of ferredoxin-NADP+
reductase. This enzyme accepts electrons, one at a time, from ferredoxin (shown in orange). (B) Ferredoxin-NADP
+ reductase first accepts one electron from reduced ferredoxin to form a flavin semiquinone intermediate. The
enzyme then accepts a second electron to form FADH2, which then transfers two electrons and a proton to NADP+ to
form NADPH.
II. Transducing and Storing Energy
19. The Light Reactions of Photosynthesis
19.4. A Proton Gradient Across the Thylakoid Membrane Drives ATP Synthesis
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 Pi. 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.
The principles by which ATP synthesis takes place in chloroplasts are nearly identical with those for 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. Such a gradient can be 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 (∆p), is described as the sum of two components: a charge gradient and a
chemical gradient. In chloroplasts, nearly all of ∆ p 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. A pH gradient of 3.5 units across the thylakoid membrane
corresponds to a proton-motive force of 0.20 V or a ∆ G of -4.8 kcal mol-1 (-20.0 kJ mol-1).
19.4.1. The ATP Synthase of Chloroplasts Closely Resembles Those of Mitochondria
and Prokaryotes
The proton-motive force generated by the light reactions is converted into ATP by the ATP synthase of chloroplasts, also
called the CF -CF complex (C stands for chloroplast and F for factor). CF1-CF0 ATP synthase closely resembles the
1
0
F1-F0 complex of mitochondria (Section 18.4.1). CF0 conducts protons across the thylakoid membrane, whereas CF1
catalyzes the formation of ATP from ADP and Pi.
CF0 is embedded in the thylakoid membrane. It consists of four different polypeptide chains known as I (17 kd), II (16.5
kd), III (8 kd), and IV (27 kd) having an estimated stoichiometry of 1:2:12:1. Subunits I, II, and III correspond to
subunits a, b, and c, respectively, of the mitochondrial F0 subunit, and subunit IV is similar in sequence to subunit a.
CF1, the site of ATP synthesis, has a subunit composition α 3 β 3 γ δ ε. The β subunits contain the catalytic sites, similar
to the F1 subunit of mitochondrial ATP synthase. Remarkably, β subunits of corn chloroplast ATP synthase 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.
Significantly, the membrane orientation of CF1-CF0 is reversed compared with that of the mitochondrial ATP synthase
(Figure 19.25). Thus, protons flow out of the thylakoid lumen through ATP synthase into the stroma. Because CF1 is on
the stromal surface of the thylakoid membrane, the newly synthesized ATP is released directly into the stromal space.
Recall that NADPH formed through the action of photosystem I and ferredoxin-NADP+ reductase also 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 CO is converted into carbohydrate.
2
19.4.2. Cyclic Electron Flow Through Photosystem I Leads to the Production of ATP
Instead of NADPH
An alternative pathway for electrons arising from P700, the reaction center of photosystem I, contributes to the
versatility of photosynthesis. The electron in reduced ferredoxin can be 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. Cyclic
photophosphorylation takes place when NADP+ is unavailable to accept electrons from reduced ferredoxin, because of a
very high ratio of NADPH to NADP+.
19.4.3. The Absorption of Eight Photons Yields One O2, Two NADPH, and Three ATP
Molecules
We can now estimate the overall stoichiometry for the light reactions. The absorption of 4 photons by photosystem II
generates 1 molecule of O2 and releases 4 protons into the thylakoid lumen. The 2 molecules of plastoquinol are
oxidized by the Q cycle of the cytochrome bf complex to release 8 protons into the lumen. Finally, the electrons from 4
molecules of reduced plastocyanin are driven to ferredoxin by the absorption of 4 additional photons. The 4 molecules of
reduced ferredoxin generate 2 molecules of NADPH. Thus, the overall reaction is:
The 12 protons released in the lumen can then flow through ATP synthase. Given the apparent stoichiometry of 12
subunit III components in CF0, we expect that 12 protons must pass through CF0 to complete one full rotation of CF1
and, hence, generate and release 3 molecules of ATP. Given this ratio, the overall reaction is
Cyclic photophosphorylation is somewhat more productive with regard to ATP synthesis. The absorption of 4 photons
by photosystem I results in the release of 8 protons into the lumen by the cytochrome bf system. These protons flow
through ATP synthase to yield 2 molecules of ATP (assuming the same ratio of ATP molecules generated per proton).
Thus, each 2 absorbed photons yield 1 molecule of ATP. No NADPH is produced.
II. Transducing and Storing Energy
19. The Light Reactions of Photosynthesis
19.4. A Proton Gradient Across the Thylakoid Membrane Drives ATP Synthesis
Figure 19.24. Jagendorf's Demonstration. Chloroplasts synthesize ATP after the imposition of a pH gradient.
II. Transducing and Storing Energy
19. The Light Reactions of Photosynthesis
19.4. A Proton Gradient Across the Thylakoid Membrane Drives ATP Synthesis
Figure 19.25. Comparison of Photosynthesis and Oxidative Phosphorylation. The light-induced electron transfer in
photosynthesis drives protons into the thylakoid lumen. The excess protons flow out of the lumen through ATP synthase
to generate ATP in the stroma. In oxidative phosphorylation, electron flow down the electron-transport chain pumps
protons out of the mitochondrial matrix. Excess protons from the intermembrane space flow into the matrix through ATP
synthase to generate ATP in the matrix.
II. Transducing and Storing Energy
19. The Light Reactions of Photosynthesis
19.4. A Proton Gradient Across the Thylakoid Membrane Drives ATP Synthesis
Figure 19.26. Cyclic Photophosphorylation. In this pathway, electrons from reduced ferredoxin are transferred to the
cytochrome bf complex rather than to ferredoxin-NADP+ reductase. The flow of electrons through cytochrome bf pumps