Voet D., Voet Ju.G. Biochemistry

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f. Plastocyanin Transports Electrons from

Cytochrome b

f to PSI

Electron transfer between cytochrome f, the terminal

electron carrier of the cytochrome b

f complex, and PSI is

mediated by plastocyanin (PC), a 99-residue, monomeric,

Cu-containing, peripheral membrane protein located on

the thylakoid luminal surface (Fig. 24-17). Thus PC is the

functional analog of cytochrome c, which transfers elec-

trons from Complex III to Complex IV in the mitochon-

drial electron-transport chain (Section 22-2C4).

PC’s redox center cycles between its Cu(I) and Cu(II)

oxidation states. The X-ray structure of PC from poplar

leaves, determined by Hans Freeman, shows that its single

Cu atom is coordinated with distorted tetrahedral geometry

by a Cys, a Met, and two His residues (Fig. 24-25). Cu(II)

complexes with four ligands normally adopt a square planar

coordination geometry, whereas those of Cu(I) are gener-

ally tetrahedral. Evidently, the strain of Cu(II)’s protein-

imposed tetrahedral coordination in PC promotes its

Section 24-2. Light Reactions 921

Figure 24-24 X-ray structure of the cytochrome b

f complex

from the thermophilic cyanobacterium Mastigocladus

laminosus. A ribbon diagram of the dimeric complex is drawn

on the left with cytochrome b

blue, subunit IV purple,

cytochrome f red, the iron–sulfur protein (ISP) yellow, and the

other subunits green.The inferred position of the lipid bilayer is

Figure 24-25 X-ray structure of plastocyanin (PC) from

poplar leaves. This 99-residue monomeric protein, a member of

the family of blue copper proteins (as is the globular domain of

Complex IV’s Subunit II, which binds the Cu

center; Section

22-2C5a), folds into a ␤ sandwich. Its Cu atom (orange sphere),

which alternates between its Cu(I) and Cu(II) oxidation states, is

tetrahedrally coordinated by the side chains of His 37, Cys 84,

His 87, and Met 92, which are shown in stick form with their C, N,

and S atoms green, blue, and yellow. Seven conserved Asp and

Glu residues (red) form a negatively charged patch on the

surface of PC that has been implicated in electrostatically binding

to a positively charged patch on the surface of cytochrome f

formed by five Lys and Arg residues. [Based on an X-ray

structure by Mitchell Guss and Hans Freeman, University of

Sydney, Australia. PDBid 1PLC.]

indicated by a yellow band. Compare this figure to Fig. 22-23

(which is upside down relative to this figure).The paths of

electron and proton transfer through the complex and the

distances, in angstroms, between redox centers are shown on the

right. [Modified from a drawing by William A. Cramer and Janet

Smith, Purdue University. PDBid 1UM3.]

JWCL281_c24_901-939.qxd 6/8/10 8:54 AM Page 921

reduction to Cu(I). This hypothesis accounts for PC’s high

standard reduction potential (0.370 V) compared to that of

the normal Cu(II)/Cu(I) half-reaction (0.158 V). This is an

example of how proteins modulate the reduction potentials

of their redox centers so as to match them to their function—

in the case of plastocyanin,the efficient transfer of electrons

from the cytochrome b

f complex to PSI.

g. PSI Resembles Both PSII and the PbRC

Cyanobacterial PSIs are trimers of protomers that each

consist of at least 11 different protein subunits coordinat-

ing ⬎100 cofactors.The X-ray structure of PSI from T. elon-

gatus (Fig. 24-26), determined at 2.5-Å resolution by Nor-

bert Krauss, Saenger, and Petra Fromme, reveals that each

of its 356-kD protomers contains nine transmembrane sub-

units (PsaA, PsbB, PsaF, PsaI–M, and PsaX) and three

stromal (cytoplasmic) subunits (PsaC–E), which collec-

tively bind 127 cofactors that comprise 30% of PSI’s mass.

The cofactors forming the PSI RC are all bound by the

homologous subunits PsaA (755 residues) and PsaB (740

residues), whose 11 transmembrane helices each are

arranged in a manner resembling those in the L and M sub-

units of the PbRC (Fig. 24-11) and the D1 and D2 subunits

of PSII (Fig. 24-19), thus supporting the hypothesis that all

RCs arose from a common ancestor. PsaA and PsaB, to-

gether with other transmembrane subunits, also bind the

cofactors of the core antenna system (see below).

Figure 24-27 indicates that PSI’s RC consists of six mol-

ecules of chlorophyll and two molecules of phylloquinone

(vitamin K

; note that it has the same phytyl side chain as

chlorophylls; Fig. 24-3),

all arranged in two pseudosymmetrically related branches,

followed by three [4Fe–4S] clusters. The primary electron

donor of this system, P700, consists of a Chl a¿ (Fig. 24-3)

and a Chl a (A1 and B1, respectively), whose rings are

(CH

)

CH H

CCH

Phylloquinone

922 Chapter 24. Photosynthesis

Figure 24-26 X-ray structure of PSI from T. elongatus.

(a) View of the trimer perpendicular to the membrane from its

stromal side. The stromal subunits have been removed for clarity.

PSI’s 3-fold axis of symmetry is represented by the small black

triangle. Different structural elements are shown for each of the

three protomers (I, II, and III). I shows the arrangement of

transmembrane helices (cylinders), which are differently colored

for each subunit.The transmembrane helices of both PsaA (blue)

and PsaB (red) are named a through k from their N- to

C-termini.The six helices in extramembranous loop regions are

drawn as spirals. II shows the transmembrane helices as cylinders

with the stromal and lumenal loop regions drawn in ribbon form.

III shows the transmembrane helices as cylinders together with

all cofactors.The RC Chl a’s and quinones, drawn in stick form,

are purple, the Fe and S atoms of the [4Fe–4S] clusters are drawn

as orange and yellow spheres, the antenna system Chl a’s (whose

side chains have been removed for clarity) are yellow, the

carotenoids are black, and the bound lipids are light green.

(b) One protomer as viewed parallel to the membrane along the

arrow in Part a with the stroma above. The transmembrane

subunits are colored as in Part a with the stromal subunits PsaC,

PsaD, and PsaE pink, cyan, and light green. The vertical line and

triangle mark the trimer’s 3-fold axis of symmetry. [Courtesy of

Wolfram Saenger, Freie Universität Berlin, Germany. PDBid

1JB0.]

(a)

(b)

JWCL281_c24_901-939.qxd 7/2/10 12:23 PM Page 922

parallel and whose Mg

2⫹

ions are separated by 6.3 Å.Thus

P700 resembles the special pair in the PbRC. However,

EPR studies indicate that ⬃80% of the unpaired electron

associated with photooxidized P700

⫹

resides on Chl a B1.

A1 is followed in the left branch of Fig. 24-27 by two more

Chl a rings, B2 and A3, and B1 is followed by A2 and B3 in

the right branch. One or both of the third pair of Chl a mol-

ecules, A3 and B3, probably form the spectroscopically

identified primary electron acceptor A

(right side of Fig.

24-18). The Mg

2⫹

ions of A3 and B3 are each axially lig-

anded by the S atom of a Met residue rather than by a His

side chain (thereby forming the only known biological ex-

amples of Mg

2⫹

¬S coordination). All of the residues in-

volved in Mg

2⫹

coordination and hydrogen bonding to

these second and third Chl a’s are strictly conserved in

PSI’s, from cyanobacteria to higher plants, thereby suggest-

ing that all of these interactions are important for fine-

tuning their redox potentials. Electrons are passed from A3

and B3 to the phylloquinones, Q

-A and Q

-B, which al-

most certainly correspond to the spectroscopically identi-

fied electron acceptor A

. Spectroscopically based kinetic

investigations indicate that, in contrast to the case for the

PbRC, electrons pass through both branches of the PSI

RC, although at different rates: 35 ⫻ 10

⫺1

for the branch

ending in Q

-B and 4.4 ⫻ 10

⫺1

for that ending in Q

-A.

Indeed, the PSI RC is most closely related to the RC of

green sulfur bacteria (a second class of photosynthetic bac-

teria), which is a true homodimer.

Up until this point, PSI’s RC resembles those of PSII

and purple photosynthetic bacteria. However, rather than

the reduced forms of either Q

-A or Q

-B dissociating

from PSI, both of these quinones directly pass their pho-

toexcited electron to a chain of three spectroscopically

identified [4Fe–4S] clusters designated F

, and F

(right side of Fig. 24-18). F

, which lies on the pseudo-2-

fold axis relating PsaA and PsaB, is coordinated by two Cys

residues from each of these subunits. F

and F

are bound

to the stromal subunit PsaC, which structurally resembles

bacterial 2[4Fe–4S] ferredoxins (e.g., Fig. 22-16). Muta-

tional studies on the Cys residues of PsaC that coordinate

its two [4Fe–4S] clusters indicate that the cluster that lies

closer to F

is F

and the more distant cluster is F

(Fig. 24-

27). The observation that both branches of PSI’s electron-

transfer pathways are active, in contrast to only one active

branch in PSII and the PbRC, is rationalized by the fact

that the two quinones at the ends of each branch are func-

tionally equivalent in PSI but functionally different in PSII

and the PbRC.

PSI’s core antenna system consists of 90 Chl a molecules

and 22 carotenoids (Fig. 24-26a). The Mg

2⫹

ions of 79 of

these Chl a molecules are axially liganded by residues of

PsaA and PsaB (mostly His side chains or protein-bound

water molecules), whereas the remaining 11 are so lig-

anded by the smaller subunits PsaJ through M and PsaX.

The spatial distribution of these antenna Chl a’s resembles

that in the core antenna subunits CP43 and CP47 of PSII.

Indeed, the N-terminal domains of PsaA and PsaB are sim-

ilar in sequence to those of CP43 and CP47 and fold into

similar structures containing six transmembrane helices

each. The carotenoids, which are mostly ␤-carotenes, are

deeply buried in the membrane, where they are in van der

Waals contact with Chl a rings. This permits efficient en-

ergy transfer from photoexcited carotenoids to Chl a as

well as protects PSI from photooxidative damage. PSI also

tightly binds four lipid molecules such that their fatty acyl

groups are embedded among the complex’s transmem-

brane helices. This strongly suggests that these lipids have

specific structural and/or functional roles rather than being

artifacts of preparation. Indeed, the head group of one of

them, a phospholipid, coordinates the Mg

2⫹

of an antenna

Chl a, an unprecedented interaction.

PSIs from higher plants are monomers rather than

trimers as are cyanobacterial PSIs. Nevertheless, the X-ray

structure of PSI from peas, determined by Nathan Nelson,

reveals that the positions and orientations of the chloro-

phylls in both species of PSIs are nearly identical, a re-

markable finding considering the ⬎1 billion years since

chloroplasts diverged from their cyanobacterial ancestors.

However, pea PSI has four antenna proteins not present in

cyanobacterial PSI that are arranged in a crescent-shaped

transmembrane belt around one side of its RC and which

collectively bind 56 chlorophyll molecules.

Section 24-2. Light Reactions 923

Figure 24-27 Cofactors of the PSI RC and PsaC. The

structure is viewed parallel to the membrane plane with the

stroma above. The Chl a and phylloquinone molecules are

arranged in two branches that are related by PSI’s 2-fold axis of

pseudosymmetry, which is vertical in this drawing. The Chl a’s are

labeled A or B to indicate that their Mg

2⫹

ions are liganded by

the side chains of PsaA or PsaB, respectively, and, from the

luminal side upward, by different colors and numbers, 1 to 3.

The phylloquinones are named Q

-A and Q

-B. PsaC is shown

in ribbon form with those portions resembling segments in

bacterial 2[4Fe–4S] ferredoxins pink and with insertions and

extensions green.The three [4Fe–4S] clusters are shown in

ball-and-stick form and labeled according to their spectroscopic

identities F

, and F

.The center-to-center distances between

cofactors (vertical black lines) are given in angstroms. Compare

this figure with Figs. 24-20 and 24-12. [Courtesy of Wolfram

Saenger, Freie Universität Berlin, Germany. PDBid 1JB0.]

JWCL281_c24_901-939.qxd 7/2/10 12:24 PM Page 923

h. PSI-Activated Electrons May Reduce NADP

ⴙ

Motivate Proton Gradient Formation

Electrons ejected from F

in PSI may follow either of two

alternative pathways (Fig. 24-18):

1. Most electrons follow a noncyclic pathway by passing

to an ⬃100-residue, single [2Fe–2S]-containing, soluble

ferredoxin (Fd) that is located in the stroma.Reduced Fd, in

turn, reduces NADP

⫹

in a reaction mediated by the ⬃310-

residue, monomeric, FAD-containing ferredoxin–NADP

ⴙ

reductase (FNR, Fig. 24-28a), to yield the final product of

the chloroplast light reaction, NADPH. Two reduced Fd

molecules successively deliver one electron each to the

FAD of FNR, which thereby sequentially assumes the neu-

tral semiquinone and fully reduced states before transfer-

ring the two electrons and a proton to the NADP

⫹

via what

is formally a hydride ion transfer.The X-ray structure of the

complex between Fd and FNR from maize leaf (Fig.24-28b),

determined by Genji Kurisu, reveals that the shortest inter-

atomic approach between Fd’s [2Fe–2S] cluster and FNR’s

FAD is the 6.0 Å between an Fe atom and FAD atom C8a

(the methyl C closest to its ribitol residue; Fig. 16-8). This is

sufficiently close for direct electron transfer through space

between these prosthetic groups. The complex is stabilized

by five salt bridges, as similarly appears to be the case for the

interaction between cytochrome f and PC.

2. Some electrons are returned from PSI, via cy-

tochrome b

, to the plastoquinone pool, thereby traversing

a cyclic pathway that translocates protons across the thy-

lakoid membrane. A mechanism that has been proposed

for this process is that Fd transfers an electron to heme x

of cytochrome b

(Fig. 24-24) rather than to FNR. Since

heme x contacts heme b

at the periphery of cytochrome

f’s Q

site, an electron injected into heme x would be ex-

pected to reduce plastoquinone via a Q cycle-like mecha-

nism (Fig. 22-31). Note that the cyclic pathway is inde-

pendent of the action of PSII and hence does not result in

the evolution of O

.This accounts for the observation that

chloroplasts absorb more than eight photons per O

mole-

cule evolved.

The cyclic electron flow presumably functions to in-

crease the amount of ATP produced relative to that of

924 Chapter 24. Photosynthesis

Figure 24-28 Ferredoxin–NADP

ⴙ

reductase. (a) The X-ray

structure of the Y308S mutant form of pea ferredoxin–NADP

⫹

reductase (FNR) in complex with FAD and NADP

⫹

.This

308-residue protein has two domains:The N-terminal domain

(gold), which forms the FAD binding site, folds into an

antiparallel ␤ barrel, whereas the C-terminal domain (magenta),

which provides the NADP

⫹

binding site, forms a dinucleotide-

binding fold (Section 8-3Bi).The FAD and NADP

⫹

are shown in

stick form with NADP

⫹

C green, FAD C cyan, N blue, O red, and

P yellow. The flavin and nicotinamide rings are in opposition

with C4 of the nicotinamide ring and C5 of the flavin ring 3.0 Å

apart, an arrangement that is consistent with direct hydride

transfer as also occurs in glutathione reductase and dihydrolipoyl

dehydrogenase (Section 21-2Ba). However, in contrast to these

latter enzymes, whose bound flavin and nicotinamide rings are

parallel, those in FNR are inclined by ⬃30°, a heretofore

unobserved binding mode. [Based on an X-ray structure by

Andrew Karplus, Cornell University. PDBid 1QFY.] (b) The

X-ray structure of the complex between Fd (red) and FNR (blue)

from maize leaf with both proteins drawn in ribbon form

embedded in their transparent solvent-accessible surfaces.The

[2Fe–2S] cluster of Fd (green) and the FAD of FNR (yellow) are

drawn in ball-and-stick form.The Fd binds in a hollow between

FNR’s two domains (Part a) such that the line joining the two

Fe’s of the [2Fe–2S] cluster lies roughly in the plane of the flavin

ring. [Courtesy of Genji Kurisu, Osaka University, Osaka, Japan.

PDBid 1GAQ.]

See Interactive Exercise 22

(a) (b)

JWCL281_c24_901-939.qxd 10/19/10 9:36 AM Page 924

NADPH and thus permits the cell to adjust the relative

amounts of these two substances produced according to its

needs. However, the mechanism that apportions electrons

between the cyclic and noncyclic pathways is unknown.

i. PSI and PSII Occupy Different Parts of

the Thylakoid Membrane

Freeze-fracture electron microscopy (Section 12-3Ca)

revealed that the protein complexes of the thylakoid mem-

brane have characteristic distributions (Fig. 24-29):

1. PSI occurs mainly in the unstacked stroma lamellae,

in contact with the stroma, where it has access to NADP

⫹

2. PSII is located almost exclusively between the

closely stacked grana, out of direct contact with the stroma.

3. Cytochrome b

f is uniformly distributed throughout

the membrane.

The high mobilities of plastoquinone and plastocyanin, the

electron carriers that shuttle electrons between these com-

plexes, permits photosynthesis to proceed at a reasonable

rate.

What function is served by the segregation of PSI and

PSII, which are typically present in chloroplasts in equimo-

lar amounts? If these two photosystems were in close prox-

imity, the higher excitation energy of PSII (P680 vs P700)

would cause it to pass a large fraction of its absorbed pho-

tons to PSI via exciton transfer; that is, PSII would act as a

light-harvesting antenna for PSI (Fig. 24-7b). The separa-

tion of these particles by around 100 Å eliminates this

difficulty.

The physical separation of PSI and PSII also permits

the chloroplast to respond to changes in illumination. The

relative amounts of light absorbed by the two photosys-

tems vary with how the light-harvesting complexes

(LHCs) are distributed between the stacked and un-

stacked portions of the thylakoid membrane. Under high

illumination (normally direct sunlight, which contains a

high proportion of short-wavelength blue light), all else

being equal, PSII absorbs more light than PSI. PSI is then

unable to take up electrons as fast as PSII can supply

them, so the plastoquinone is predominantly in its reduced

state. The reduced plastoquinone activates a protein ki-

nase to phosphorylate specific Thr residues of the LHCs,

which, in response, migrate to the unstacked regions of the

thylakoid membrane, where they bind to PSI. A greater

fraction of the incident light is thereby funneled to PSI.

Under low illumination (normally shady light, which con-

tains a high proportion of long-wavelength red light), PSI

takes up electrons faster than PSII can provide them so

that plastoquinone predominantly assumes its oxidized

form. The LHCs are consequently dephosphorylated and

migrate to the stacked portions of the thylakoid mem-

brane, where they drive PSII. The chloroplast therefore

maintains the balance between its two photosystems by a

light-activated feedback mechanism.

D. Photophosphorylation

Chloroplasts generate ATP in much the same way as mito-

chondria, that is, by coupling the dissipation of a proton gra-

dient to the enzymatic synthesis of ATP (Section 22-3C).

This was clearly demonstrated by the imposition of an arti-

ficially produced pH gradient across the thylakoid mem-

brane. Chloroplasts were soaked, in the dark, for several

hours in a succinic acid solution at pH 4 so as to bring the

thylakoid lumen to this pH (the thylakoid membrane is

permeable to unionized succinic acid). The abrupt transfer

of these chloroplasts to an ADP ⫹ P

–containing buffer at

pH 8 resulted in an impressive burst of ATP synthesis:

About 100 ATPs were synthesized per molecule of

cytochrome f present. Moreover, the amount of ATP

Section 24-2. Light Reactions 925

Figure 24-29 Segregation of PSI and PSII. The distribution of

photosynthetic protein complexes between the stacked (grana)

and the unstacked (stroma exposed) regions of the thylakoid

ATP synthase

Unstacked membranes

(stromal lamellae)

Stacked

membranes

(grana)

PSI complex

PSII complex

Cytochrome b

membrane is shown. [After Anderson, J.M. and Anderson, B.,

Trends Biochem. Sci. 7, 291 (1982).]

JWCL281_c24_901-939.qxd 3/25/10 12:05 PM Page 925

synthesized was unaffected by the presence of electron-

transport inhibitors such as DCMU. This, together with the

observations that photophosphorylation requires an intact

thylakoid membrane and that proton translocators such as

2,4-dinitrophenol (Section 22-3D) uncouple photophos-

phorylation from light-driven electron transport, provides

convincing evidence favoring the chemiosmotic hypothesis

(Section 22-3A).

a. Chloroplast Proton-Translocating ATP Synthase

Resembles That of Mitochondria

Electron micrographs of thylakoid membrane stromal

surfaces and bacterial plasma membrane inner surfaces re-

veal lollipop-shaped structures (Fig. 24-30). These closely

resemble the F

units of the proton-translocating ATP syn-

thase studding the matrix surfaces of inner mitochondrial

membranes (Fig. 22-36a). In fact, the chloroplast ATP syn-

thase, which is termed the CF

complex (C for chloro-

plast), has remarkably similar properties to the mitochon-

drial F

complex (Section 22-3C). For example,

1. Both F

and CF

units are hydrophobic transmem-

brane proteins that contain a proton-translocating channel.

2. Both F

and CF

are hydrophilic peripheral mem-

brane proteins of subunit composition ␣

␤

␥␦ε, of which ␤

is a reversible ATPase.

3. Both ATP synthases are inhibited by oligomycin.

4. Chloroplast ATP synthase translocates protons out

of the thylakoid lumen into the stroma (Fig. 24-17), and

mitochondrial ATP synthase conducts them into the matrix

space (the mitochondrial equivalent of the stroma) from

the intermembrane space (Section 22-3A).

Clearly, proton-translocating ATP synthases must have

evolved very early in the history of cellular life. Chloroplast

ATP synthase is located in the unstacked portions of the

thylakoid membrane, in contact with the stroma, where

there is room for the bulky CF

globule and access to ADP

(Fig. 24-29).

b. Photosynthesis with Noncyclic Electron

Transport Produces Around 1.25 ATP

Equivalents per Absorbed Photon

At saturating light intensities, chloroplasts generate

proton gradients of ⬃3.5 pH units across their thylakoid

membranes. This, as we have seen (Figs. 24-17 and 24-18),

arises from two sources:

1. The evolution of a molecule of O

from two H

molecules releases four protons into the thylakoid lumen.

These protons should be considered as being supplied from

the stroma by the protons and H atoms taken up in the syn-

thesis of NADPH.

2. The transport of the liberated four electrons through

the cytochrome b

f complex occurs with the translocation

of what is estimated to be eight protons from the stroma to

the thylakoid lumen.

Altogether ⬃12 protons are translocated per molecule of O

produced by noncyclic electron transport.

The thylakoid membrane, in contrast to the inner mito-

chondrial membrane, is permeable to ions such as Mg

2⫹

and Cl

⫺

. Translocation of protons and electrons across the

thylakoid membrane is consequently accompanied by the

passage of these ions so as to maintain electrical neutrality

(Mg

2⫹

out and Cl

⫺

in). This all but eliminates the mem-

brane potential, ⌬⌿ (Eq. [22.1]). The electrochemical gradi-

ent in chloroplasts is therefore almost entirely a result of the

pH gradient.

Chloroplast ATP synthase, according to most estimates,

produces one ATP for every three protons it transports out

of the thylakoid lumen. Noncyclic electron transport in

chloroplasts therefore results in the production of

⬃12/3 ⫽ 4 molecules of ATP per molecule of O

evolved

(although this quantity is subject to revision) or around

half an ATP per photon absorbed. Cyclic electron trans-

port is a more productive ATP generator since it yields

two-thirds of an ATP (two protons transported) per ab-

sorbed photon.The noncyclic process, of course, also yields

NADPH, each molecule of which has the free energy to

produce three ATPs (Section 22-2A; although this does not

normally occur), for a total of six more ATP equivalents

per O

produced. Consequently, the energetic efficiency of

the noncyclic process is 4兾8 ⫹ 6兾8 ⫽ 1.25 ATP equivalents

per absorbed photon.

3 DARK REACTIONS

In the previous section we saw how light energy is har-

nessed to generate ATP and NADPH. In this section we

discuss how these products are used to synthesize carbohy-

drates and other substances from CO

926 Chapter 24. Photosynthesis

Figure 24-30 Electron micrograph of thylakoids. The CF

“lollipops” of their ATP synthases project from their stromal

surfaces. Compare this with Fig. 22-36a. [Courtesy of Peter

Hinkle, Cornell University.]

JWCL281_c24_901-939.qxd 3/25/10 12:05 PM Page 926

A. The Calvin Cycle

The metabolic pathway by which plants incorporate CO

into carbohydrates was elucidated between 1946 and 1953

by Melvin Calvin, James Bassham, and Andrew Benson.

They did so by tracing the metabolic fate of the radioactive

label from

as it passed through a series of photosyn-

thetic intermediates. The basic experimental strategy they

used was to expose growing cultures of algae, such as

Chlorella, to

for varying times and under differing il-

lumination conditions and then to drop the cells into boil-

ing alcohol so as to disrupt them while preserving their la-

beling pattern.The radioactive products were subsequently

separated and identified (an often difficult task) through

the use of the then recently developed technique of two-

dimensional paper chromatography (Section 6-3Dc) coupled

with autoradiography.The overall pathway, diagrammed in

Fig. 24-31, is known as the Calvin cycle or the reductive

pentose phosphate cycle.

Some of Calvin’s earliest experiments indicated that al-

gae exposed to

for a minute or more had synthesized

a complex mixture of labeled metabolic products, including

sugars and amino acids. By inactivating the algae within 5 s

of their exposure to

, however, it was shown that the

first stable radioactively labeled compound formed is 3-

phosphoglycerate (3PG), which is initially labeled only in its

carboxyl group. This result immediately suggested, in anal-

ogy with most biochemical experience, that the 3PG was

formed by the carboxylation of a C

compound. Yet the

failure to find any such precursor eventually forced this hy-

pothesis to be abandoned. The actual carboxylation reac-

tion was discovered through an experiment in which illu-

minated algae had been exposed to

for ⬃10 min so

that the levels of their labeled photosynthetic intermedi-

ates had reached a steady state. The CO

was then with-

drawn. As expected, the carboxylation product, 3PG, de-

creased in concentration (Fig. 24-32) because it was

depleted by reactions farther along the pathway. The con-

centration of ribulose-5-phosphate (Ru5P),

however, simultaneously increased. Evidently, Ru5P is the

Calvin cycle’s carboxylation substrate. If so, the resulting

carboxylation product must split into two C

com-

pounds, one of which is 3PG (Fig. 24-31, Reaction 2). A

consideration of the oxidation states of Ru5P and CO

in-

dicates that, in fact, both C

compounds must be 3PG and

that the carboxylation reaction requires no external redox

source.

While the search for the carboxylation substrate was go-

ing on, several other photosynthetic intermediates had

Ribulose-5-phosphate (Ru5P)

HCOH

OPO

–

been identified and, through chemical degradation studies,

their labeling patterns had been elucidated. For example,

the hexose fructose-1,6-bisphosphate (FBP) is initially la-

beled only at its C3 and C4 positions (Fig. 24-31) but later

becomes labeled to a lesser degree at its other atoms. Sim-

ilarly, a series of tetrose, pentose, hexose, and heptose phos-

phates were isolated that had the identities and initial la-

beling patterns indicated in Fig. 24-31. A consideration of

the flow of the labeled atoms through these various inter-

mediates led, in what was a milestone of metabolic bio-

chemistry, to the deduction of the Calvin cycle as is dia-

grammed in Fig. 24-31. The existence of many of its

postulated reactions was eventually confirmed by in vitro

studies using purified enzymes.

a. The Calvin Cycle Generates GAP from

via a Two-Stage Process

The Calvin cycle may be considered to have two stages:

Stage 1 The production phase (top line of Fig. 24-31), in

which three molecules of Ru5P react with three molecules

of CO

to yield six molecules of glyceraldehyde-3-phosphate

(GAP) at the expense of nine ATP and six NADPH mole-

cules. The cyclic nature of the pathway makes this process

equivalent to the synthesis of one GAP from three CO

molecules. Indeed, at this point, one GAP can be bled off

from the cycle for use in biosynthesis (see Stage 2).

Stage 2 The recovery phase (bottom lines of Fig. 24-31),

in which the carbon atoms of the remaining five GAPs are

shuffled in a remarkable series of reactions, similar to those

of the pentose phosphate pathway (Section 23-4), to re-

form the three Ru5Ps with which the cycle began. Indeed,

the elucidation of the pentose phosphate pathway at about

the same time that the Calvin cycle was being worked out

provided much of the biochemical evidence in support of

the Calvin cycle. This stage can be conceptually decom-

posed into four sets of reactions (with the numbers keyed

to the corresponding reactions in Fig. 24-31):

6. C

⫹ C

8. C

⫹ C

9. C

⫹ C

11. C

⫹ C

The overall stoichiometry for this process is therefore

Note that this stage of the Calvin cycle occurs without fur-

ther input of free energy (ATP) or reducing power

(NADPH).

b. Most Calvin Cycle Reactions Also Occur in

Other Metabolic Pathways

The types of reactions in the Calvin cycle are all familiar

(Section 23-4), with the exception of the carboxylation

reaction. This first stage of the Calvin cycle begins with

the phosphorylation of Ru5P by phosphoribulokinase to

form ribulose-1, 5-bisphosphate (RuBP). Following the

Section 24-3. Dark Reactions 927

JWCL281_c24_901-939.qxd 3/25/10 12:05 PM Page 927

928 Chapter 24. Photosynthesis

ribulose

bisphos-

phate

carboxylase

phospho-

glycerate

kinase

triose phosphate

isomerase

aldolase

Ribulose-

1,5-bis-

phosphate

(RuBP)

3-Phospho-

glycerate

(3PG)

1,3-Bisphospho-

glycerate

(BPG)

Glyceraldehyde-

3-phosphate

(GAP)

phospho-

ribulo-

kinase

ATP

fructose

bisphosphatase

phosphopentose

epimerase

transketolase

ADP

ATP ADP

OPO

2–

OPO

2–

COHH

OPO

2–

OPO

2–

OPO

2–

HHO

–

Ribulose-5-

phosphate

(Ru5P)

OPO

2–

C OHH

COHH

glyceraldehyde-

3-phosphate

dehydrogenase

NADPH NADP

+ P

CCHO

COHH

OPO

2–

COHH

OPO

2–

Ribulose-5-

phosphate

(Ru5P)

Dihydroxyacetone

phosphate

(DHAP)

OPO

2–

C OHH

COHH

Fructose-1,6-

bisphosphate

(FBP)

Sedoheptulose-1,7-

bisphosphate (SBP)

Sedoheptulose-7-

phosphate (S7P)

OPO

2–

OPO

2–

C OHH

COHH

OPO

2–

C OHH

OPO

2–

COHH

C OHH

OPO

2–

COHH

Fructose-6-

phosphate

(F6P)

Xylulose-5-

phosphate

(Xu5P)

Ribose-5-

phosphate

(R5P)

OPO

2–

C OHH

COHH

C OHH

OPO

2–

COHH

CHO

OPO

2–

COHH

OPO

2–

Products

Erythrose-4-

phosphate (E4P)

sedoheptulose

bisphosphatase

CHO

OPO

2–

C OHH

COHH

ribose

phosphate

isomerase

11 transketolase

JWCL281_c24_901-939.qxd 3/25/10 12:05 PM Page 928

carboxylation step, which is discussed below, the resulting

3PG is converted first to 1,3-bisphosphoglycerate (BPG)

and then to GAP. This latter sequence is the reverse of

two consecutive glycolytic reactions (Sections 17-2G and

17-2F) except that the Calvin cycle reaction involves

NADPH rather than NADH.

The second stage of the Calvin cycle begins with the re-

verse of a familiar glycolytic reaction, the isomerization of

GAP to dihydroxyacetone phosphate (DHAP) by triose

phosphate isomerase (Section 17-2E). Following this,

DHAP is directed along two analogous paths (Fig. 24-31):

Reactions 6–8 or Reactions 9–11. Reactions 6 and 9 are

aldolase-catalyzed aldol condensations in which DHAP is

linked to an aldehyde (aldolase is specific for DHAP but

accepts a variety of aldehydes). Reaction 6 is also the re-

verse of a glycolytic reaction (Section 17-2D). Reactions 7

and 10 are phosphate hydrolysis reactions that are cat-

alyzed, respectively, by fructose bisphosphatase (FBPase,

which we previously encountered in our discussion of

glycolytic substrate cycles and gluconeogenesis; Sections

17-4Ff and 23-1Ah) and sedoheptulose bisphosphatase

(SBPase). The remaining Calvin cycle reactions are cat-

alyzed by enzymes that also participate in the pentose phos-

phate pathway. In Reactions 8 and 11, both catalyzed by

transketolase, a C

keto unit (shaded in green in Fig. 24-31)

is transferred from a ketose to GAP to form xylulose-

5-phosphate (Xu5P) and leave the aldoses erythrose-

4-phosphate (E4P) in Reaction 8 and ribose-5-phosphate

(R5P) in Reaction 11. The E4P produced by Reaction 8

feeds into Reaction 9. The Xu5Ps produced by Reactions 8

and 11 are converted to Ru5P by phosphopentose

epimerase in Reaction 12.The R5P from Reaction 11 is also

converted to Ru5P by ribose phosphate isomerase in Reac-

tion 13, thereby completing a turn of the Calvin cycle. Thus

only 3 of the 11 Calvin cycle enzymes, phosphoribulokinase,

the carboxylation enzyme ribulose bisphosphate carboxy-

lase, and SBPase, have no equivalents in animal tissues.

c. RuBP Carboxylase Catalyzes CO

Fixation

in an Exergonic Process

The enzyme that catalyzes CO

fixation, ribulose bis-

phosphate carboxylase (RuBP carboxylase), is arguably

the world’s most important enzyme, since nearly all life on

Earth ultimately depends on its action. This protein, pre-

sumably as a consequence of its particularly low catalytic

efficiency (k

cat

⫽ ⬃3 s

⫺1

), comprises up to 50% of leaf pro-

teins and is therefore the most abundant protein in the

biosphere (it is estimated to be synthesized at the rate of

⬃4 ⫻ 10

tons/year, which fixes ⬃10

tons of CO

/year; in

comparison crude oil is consumed at the rate of ⬃3 ⫻ 10

tons/year). RuBP carboxylase from higher plants and most

photosynthetic microorganisms consists of eight large (L)

subunits (477 residues in tobacco leaves) encoded by

chloroplast DNA and eight small (S) subunits (123

residues) specified by a nuclear gene (the RuBP carboxy-

lase from certain photosynthetic bacteria is an L

dimer

whose L subunit has 28% sequence identity with and is

structurally similar to that of the L

enzyme). X-ray stud-

ies by Carl-Ivar Brändén and by David Eisenberg demon-

strated that the L

enzyme has the D

symmetry of a square

prism (Fig. 24-33a,b). The L subunit contains the enzyme’s

catalytic site, as is demonstrated by its enzymatic activity in

the absence of the S subunit. It consists of two domains

(Fig. 24-33c): Residues 1 to 150 form a mixed five-stranded

␤ sheet and residues 151 to 477 fold into an ␣/␤ barrel (Fig.

8-19b,c) which, as do nearly all known ␣/␤ barrel enzymes

(Section 8-3Bh), contains the enzyme’s active site at the

mouth of the barrel near the C-terminus of its ␤ strands.

The function of the S subunit is unknown; attempts to show

that it has a regulatory role, in analogy with other enzymes,

have been unsuccessful.

Section 24-3. Dark Reactions 929

Figure 24-31 The Calvin cycle. (Opposite) The number of

lines in an arrow indicates the number of molecules reacting in

that step for a single turn of the cycle that converts three CO

molecules to one GAP molecule. For the sake of clarity, the

sugars are all shown in their linear forms, although the hexoses

and heptoses predominantly exist in their cyclic forms (Section

11-1B).The

C-labeling patterns generated in one turn of the

cycle through the use of

are indicated in red. Note that two

of the Ru5Ps are labeled only at C3, whereas the third Ru5P is

equally labeled at C1, C2, and C3.

See the Animated Figures

Figure 24-32 Algal 3PG and RuBP levels on removal of CO

The time course of the levels of 3PG (purple curve) and RuBP

(green curve) in steady-state

-labeled, illuminated algae is

shown during a period in which the CO

(orange curve) is

abruptly withdrawn. In the absence of CO

, the 3PG

concentration rapidly decreases because it is taken up by the

reactions of the Calvin cycle but cannot be replenished by them.

Conversely, the RuBP concentration transiently increases as it is

synthesized from the residual pool of Calvin cycle intermediates

but, in the absence of CO

, cannot be used for their regeneration.

01234567

Reservoir size

Time (min)

withdrawal

RuBP

3PG

JWCL281_c24_901-939.qxd 6/16/10 12:04 PM Page 929

The accepted mechanism of RuBP carboxylase, which

was largely formulated by Calvin, is indicated in Fig. 24-34.

Abstraction of the C3 proton of RuBP, the reaction’s rate-

determining step, generates an enediolate that nucleophili-

cally attacks CO

(not HCO

⫺

). The resulting ␤-keto acid is

rapidly attacked at its C3 position by H

O to yield an

adduct that splits, by a reaction similar to aldol cleavage, to

yield the two product 3PG molecules. The following evi-

dence favors this mechanism:

1. The C3 proton of enzyme-bound RuBP exchanges

with solvent, an observation compatible with the existence

of the enediolate intermediate.

2. The C2 and C3 oxygen atoms remain attached to

their respective C atoms, which eliminates mechanisms in-

volving a covalent adduct such as a Schiff base between

RuBP and the enzyme.

3. The trapping of the proposed ␤-keto acid intermedi-

ate by borohydride reduction and the tight enzymatic bind-

ing of its analogs, such as 2-carboxyarabinitol-1-phosphate

(CA1P) and 2-carboxyarabinitol-1,5-bisphosphate (CABP),

provide strong evidence for the existence of this intermediate.

The driving force for the overall reaction, which is highly

exergonic (⌬G°¿ ⫽⫺35.1 kJ ⴢ mol

⫺1

), is provided by the

2-Carboxyarabinitol-

1-phosphate

(CA1P)

HCOH

HO C CO

OPO

–

2-Carboxyarabinitol-

1,5-bisphosphate

(CABP)

HCOH

HO C CO

OPO

–

OPO

–

930 Chapter 24. Photosynthesis

Figure 24-33 X-ray structure of tobacco RuBP carboxylase in

complex with the transition state inhibitor 2-carboxyarabinitol-

1,5-bisphosphate. The ⬃535-kD L

protein has D

symmetry

(the rotational symmetry of a square prism; Fig. 8-65b).

(a) Surface diagram viewed along the protein’s 4-fold axis, and

(b) along a 2-fold axis. Parts a and b are related by a 90° rotation

about the horizontal axis.The elongated L subunits can be

considered to associate as two interdigitated tetramers, with that

extending from the top in Part b cyan and that extending from

the bottom green.The S subunits, which form C

tetramers that

cap the top and bottom of the complex, are alternately yellow

and orange. The transition state inhibitor 2-carboxyarabinitol-

1,5-bisphosphate (CABP; see text), which is partially visible in

Part b, is drawn in stick form with C green, O red, and P orange.

green subunit in Part b and colored in rainbow order from its

N-terminus (blue) to its C-terminus (red).The 2-carboxyarabinitol-

1,5-bisphosphate is bound in the mouth of the enzyme’s ␣/␤

barrel. [Based on an X-ray structure by David Eisenberg, UCLA.

PDBid 1RLC.]

(a)

(b)

(c)

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