Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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of the structure and absorb light maximally at 700 nm. This center, P700, initiates photoinduced charge separation

(Figure 19.20). The electron is transferred down a pathway through chlorophyll at site A

and quinone at site A

to a set

of 4Fe-4S clusters. From there, the electron is transferred to ferredoxin (Fd), a soluble protein containing a 2Fe-2S

cluster coordinated to four cysteine residues (Figure 19.21). The positive charge of P700

is neutralized by the transfer

of an electron from reduced plastocyanin. Thus, the overall reaction catalyzed by photosystem I is a simple one-electron

oxidation-reduction reaction.

Given that the reduction potentials for plastocyanin and ferredoxin are +0.37 V and -0.45 V, respectively, the standard

free energy for the oxidation of reduced plastocyanin by oxidized ferredoxin is +18.9 kcal mol

-1

(+79.1 kJ mol

-1

). This

uphill reaction is driven by the absorption of a 700-nm photon which has an energy of 40.9 kcal mol

-1

(171 kJ mol

-1

The cooperation between photosystem I and photosystem II creates electron flow from H

O to NADP

. The pathway of

electron flow is called the Z scheme of photosynthesis because the redox diagram from P680 to P700* looks like the

letter Z (Figure 19.22).

19.3.4. Ferredoxin-NADP

Reductase Converts NADP

into NADPH

Although reduced ferredoxin is a strong reductant, it is not useful for driving many reactions, in part because ferredoxin

carries only one available electron. In contrast, NADPH, a two-electron reductant, is widely used in biosynthetic

processes, including the reactions of the Calvin cycle (Chapter 20). How can reduced ferredoxin be used to drive the

reduction of NADP

to NADPH? This reaction is catalyzed by ferredoxin-NADP

reductase, a flavoprotein (Figure

19.23A). The bound FAD moiety in this enzyme accepts electrons, one at a time, from two molecules of reduced

ferredoxin as it proceeds from its oxidized form, through a semiquinone intermediate, to its fully reduced form (Figure

19.23B). The enzyme then transfers a hydride ion to NADP

to form NADPH. Note that this reaction takes place on the

stromal side of the membrane. Hence, the uptake of a proton in the reduction of NADP

further contributes to the proton

gradient across the thylakoid membrane.

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 19.11. Two Photosystems. The absorption of photons by two distinct photosytems (PS I and PS II) is required

for complete electron flow from water to NADP

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 19.12. The Structure of Photosystem II.

The D1 and D2 subunits are shown in red and blue and the structure of

a bound cytochrome molecule is shown in yellow. Chlorophyll molecules are shown in green.

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 19.13. Electron Flow Through Photosystem II. Light absorption induces electron transfer from P680 down an

electron-transfer pathway to an exchangeable plastoquinone. The positive charge on P680 is neutralized by electron flow

from water molecules bound at the manganese center.

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 19.14. Four Photons Are Required to Generate One Oxygen Molecule. When dark-adapted chloroplasts are

exposed to a brief flash of light, one electron passes through photosystem II. Monitoring the O

released after each flash

reveals that four flashes are required to generate each O

molecule. The peaks in O

release occur after the 3rd, 7th, and

11th flashes because the dark-adapted chloroplasts start in the S

state-that is, the one-electron reduced state.

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 19.15. A Plausible Scheme for Oxygen Evolution from the Manganese Center. A possible partial structure

for the manganese center is shown. The center is oxidized, one electron at a time, until two bound H

O molecules are

linked to form a molecule of O

, which is then released from the center. A tyrosine residue (not shown) also participates

in the coupled proton-electron transfer steps. The structures are designated S

through S

to indicate the number of

electrons that have been removed.

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 19.16. Proton-Gradient Direction. Photosystem II releases protons into the thylakoid lumen and takes them up

from the stroma. The result is a pH gradient across the thylakoid membrane with an excess of protons (low pH) inside.

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 19.17. Structure of Plastocyanin.

Two histidine residues, a cysteine residue, and a methionine residue

coordinate a copper ion in a distorted tetrahedral manner in this protein, which carries electrons from cytochrome

bf complex to photosystem I.

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 19.18. Cytochrome BF Contribution to Proton Gradient. The cytochrome bf complex oxidizes QH

to Q

through the Q cycle. Four protons are released into the thylakoid lumen in each cycle.

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 19.19. Structure of Photosystem I.

The psaA and psaB subunits are shown in yellow, with the regions similar to

those in the core of photosytem II shown in red and blue. Chlorophyll molecules are shown in green and three 4Fe-

4S clusters are indicated.

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 19.20. Electron Flow Through Photosystem I to Ferredoxin. Light absorption induces electron transfer from

P700 down an electron-transfer pathway that includes a chlorophyll molecule, a quinone molecule, and three 4Fe-4S

clusters to reach ferredoxin. The positive charge left on P700 is neutralized by electron transfer from reduced

plastocyanin.

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 19.21. Structure of Ferredoxin. In plants, ferredoxin contains a 2Fe-2S cluster. This protein accepts electrons

from photosystem I and carries them to ferredoxin-NADP

reductase.

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 19.22. Pathway of Electron Flow From H

O to NADP

in Photosynthesis. This endergonic reaction is made

possible by the absorption of light by photosystem II (P680) and photosystem I (P700). Abbreviations: Ph, pheophytin;

and Q

, plastoquinone-binding proteins; Pc, plastocyanin; A

and A

, acceptors of electrons from P700*; Fd,

ferredoxin; Mn, manganese.

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 FADH

, which then transfers two electrons and a proton to NADP

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 P

. 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 Mg

. The light-induced transfer of H

into the thylakoid space is accompanied by the transfer of

either Cl

in the same direction or Mg

(1 Mg

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). CF

-CF

ATP synthase closely resembles the

-F

complex of mitochondria (Section 18.4.1). CF

conducts protons across the thylakoid membrane, whereas CF

catalyzes the formation of ATP from ADP and P

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 F

subunit, and subunit IV is similar in sequence to subunit a.

, the site of ATP synthesis, has a subunit composition α

γ δ ε. The β subunits contain the catalytic sites, similar

to the F

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 CF

-CF

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 CF

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.

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 O

is not formed from H

O. 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 O

, 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 O

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 CF

, we expect that 12 protons must pass through CF

to complete one full rotation of CF

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