Voet D., Voet Ju.G. Biochemistry

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associates with the M subunit BPheo b (Fig. 24-12a, left).

These various chromophores are closely associated with a

number of protein aromatic rings, which are therefore also

thought to participate in the electron-transfer process

described below. The Fe(II) is positioned between the

menaquinone and ubiquinone rings and is octahedrally lig-

anded by four His side chains and the two carboxyl oxygen

atoms of a Glu side chain. Curiously, the two symmetry re-

lated groups of chromophores are not functionally equiva-

lent; electrons, as we shall see, are almost exclusively trans-

ferred through the L subunit (the right sides of Figs. 24-11

and 24-12). This effect is generally attributed to subtle

structural and electronic differences between the L and M

subunits.

c. The Electronic States of Molecules Undergoing

Fast Reactions Can Be Monitored by EPR and

Laser Spectroscopy Techniques

The turnover time of a photosynthetic reaction cycle, as

we have seen, is only a few milliseconds. Its sequence of re-

actions can therefore only be traced by measurements that

can follow extremely rapid electronic changes in mole-

cules. Two techniques are well suited to this task:

1. Electron paramagnetic resonance (EPR) spectroscopy

[also called electron spin resonance (ESR) spectroscopy],

which detects the spins of unpaired electrons in a manner

analogous to the detection of nuclear spins in NMR spec-

troscopy. A molecular species with unpaired electrons,

such as an organic radical or a transition metal ion, has a

characteristic EPR spectrum because its unpaired elec-

trons interact with the magnetic fields generated by the

nuclei and the other electrons of the molecule. Paramag-

netic species as short lived as 10 ps can exhibit definitive

EPR spectra.

2. Optical spectroscopy using pulsed lasers. Laser flashes

as brief as 20 attoseconds (as; 1 as ⫽ 10

⫺18

s) have been

generated. By monitoring the bleaching (disappearance) of

certain absorption bands and the emergence of others,

laser spectroscopy can track the time course of a fast reac-

tion process.

Section 24-2. Light Reactions 911

Fe(II)

0 s(a) Special pair

Menaquinone (Q

)

Ubiquinone (Q

)

BPheo b

Accessory BChl b

hν

3 × 10

–12

s(b)

200 × 10

–12

s(c)

100 × 10

–6

s(d)

BPheo b

Figure 24-12 Sequence of excitations in the bacterial RC of

Rps. viridis. The RC chromophores are shown in the same view

as in Fig. 12-26a, which resembles that in Fig. 24-11a. Note that

their rings, but not their aliphatic side chains, are arranged with

close to 2-fold symmetry. (a) At zero time, a photon is absorbed

by the “special pair” of BChl b molecules, thereby collectively

raising them to an excited state [in each step, the excited

molecule(s) is shown in red]. (b) Within 3 ps, an excited electron

has passed to the BPheo b of the L subunit (right arm of the

system) without becoming closely associated with the accessory

BChl b.The special pair is thereby left with a positive charge.

menaquinone (Q

, which is ubiquinone in Rb. sphaeroides).

(d) Within the next 100 ␮s, the special pair has been reduced (via

an electron-transport chain discussed in the text), thereby

eliminating its positive charge, while the excited electron

migrates to the ubiquinone (Q

).After a second such electron

has been transferred to Q

, it picks up two protons from solution

and exchanges with the membrane-bound ubiquinone pool.

See Kinemage Exercise 8-2

JWCL281_c24_901-939.qxd 6/8/10 8:54 AM Page 911

d. Photon Absorption Rapidly Photooxidizes

the Special Pair

The sequence of photochemical events mediated by the

photosynthetic reaction center is diagrammed in Fig. 24-12:

(a) The primary photochemical event of bacterial photo-

synthesis is absorption of a photon by the special pair (P870

or P960 depending on whether it consists of BChl a or b;

here, for argument’s sake, we assume it to be P960). This

event is nearly instantaneous;it occupies the ⬃3-fs oscillation

time of a light wave. EPR measurements established that

P960 is, in fact, a pair of BChl b molecules and indicated that

the excited electron is delocalized over both of them.

(b) P960*, the excited state of P960, has but a fleeting

existence. Laser spectroscopy has demonstrated that

within ⬃3 ps after its formation, P960* has transferred an

electron to the BPheo b on the right in Fig. 24-12b to yield

P960

⫹

BPheo b

⫺

. In forming this radical pair, the trans-

ferred electron must pass near but seems not to reduce the

intervening BChl b (which is therefore termed an acces-

sory chlorophyll), although its position strongly suggests

that it has an important role in conveying electrons.

grated to the menaquinone (or,in many species, the second

ubiquinone), designated Q

, to form the anionic semi-

quinone radical All these electron transfers, as dia-

grammed in Fig. 24-13, are to progressively lower energy

states, which makes this process all but irreversible.

Rapid removal of the excited electron from the vicinity of

P960

⫹

is an essential feature of the PbRC; this prevents

back reactions that would return the electron to P960

⫹

as to provide the time required for the wasteful internal

conversion of its excitation energy to heat. In fact, this se-

quence of electron transfers is so efficient that its overall

quantum yield (ratio of molecules reacted to photons ab-

sorbed) is virtually 100%. No man-made device has yet ap-

proached this level of efficiency.

e. Electrons Are Returned to the Photooxidized

Special Pair via an Electron-Transport Chain

The remainder of the photosynthetic electron-transport

process occurs on a much slower timescale.Within ⬃100 ␮s

after its formation, , which occupies a hydrophobic

pocket in the protein, transfers its excited electron to the

more solvent-exposed ubiquinone, Q

, to form (Fig.

24-12d). The nonheme Fe(II) is not reduced in this

process and, in fact, its removal only slightly affects the

electron transfer rate, so that the Fe(II) probably func-

tions to fine-tune the PbRC’s electronic character. Q

never becomes fully reduced; it shuttles between its oxi-

dized and semiquinone forms. Moreover, the lifetime of

is so short that it never becomes protonated. In con-

trast, once the PbRC again becomes excited, it transfers a

second electron to to form the fully reduced This

anionic quinol takes up two protons from the solution on

the cytoplasmic side of the plasma membrane to form

.Thus Q

is a molecular transducer that converts two

2⫺

⫺

ⴢ

⫺

ⴢ

⫺

ⴢ

⫺

ⴢ

⫺

ⴢ

light-driven one-electron excitations to a two-electron

chemical reduction.

The electrons taken up by Q

are eventually returned

to P960

⫹

via a complex electron-transport chain (Fig. 24-13).

The details of this process are more species dependent than

the preceding and are not so well understood. The avail-

able redox carriers include a membrane-bound pool of

ubiquinone molecules, cytochrome bc

, and cytochrome c

Cytochrome bc

is a transmembrane protein complex

composed of a [2Fe–2S] cluster–containing subunit; a heme

c-containing cytochrome c

; a cytochrome b that contains

two functionally inequivalent heme b’s, b

and b

(H and L

for high and low potential); and, in some species, a fourth

subunit. Note that cytochrome bc

is strikingly similar to

the proton-translocating Complex III of mitochondria

(Section 22-2C3a), which is also called cytochrome bc

.The

electron-transport pathway leads from Q

on the cyto-

plasmic side of the plasma membrane, through the

ubiquinone pool, with which Q

exchanges, to cy-

tochrome bc

, and then to cytochrome c

on the external

(periplasmic) side of the plasma membrane. The reduced

cytochrome c

, which, as its name implies, closely resembles

mitochondrial cytochrome c, diffuses along the external mem-

brane surface until it reacts with the membrane-spanning

PbRC to transfer an electron to P960

⫹

(the structures of

several c-type cytochromes, including that of cytochrome c

from Rs. rubrum, are diagrammed in Fig. 9-41). In Rps.

viridis, the four-heme c-type cytochrome bound to the

PbRC complex on the external side of the plasma mem-

brane (Fig. 12-26) is interposed between cytochrome c

and

P960

⫹

. Note that one of this c-type cytochrome’s hemes is

positioned to reduce the photooxidized special pair. The

PbRC is thereby prepared to absorb another photon.

f. Photosynthetic Electron Transport Drives the

Formation of a Proton Gradient

Since electron transport in PbRCs is a cyclic process (Fig.

24-13), it results in no net oxidation–reduction. Rather, it func-

tions to translocate the cytoplasmic protons acquired by Q

across the plasma membrane, thereby making the cell alkaline

relative to its environment. The mechanism of this process is

essentially identical to that of proton transport in mitochon-

drial Complex III (Section 22-3Be); that is, in addition to the

translocation of the two H

⫹

resulting from the two-electron

reduction of Q

to QH

, a Q cycle mediated by cytochrome

translocates two H

⫹

for a total of four H

⫹

translocated per

two photons absorbed (Fig. 24-13a; also see Fig. 22-31). Syn-

thesis of ATP, a process known as photophosphorylation, is

driven by the dissipation of the resulting pH gradient in a man-

ner that closely resembles ATP synthesis in oxidative phospho-

rylation (Section 22-3C). We further discuss the mechanism of

photophosphorylation in Section 24-2D.

Photosynthetic bacteria use photophosphorylation-

generated ATP to drive their various endergonic processes.

However, unlike cyanobacteria and plants, which generate

their required reducing equivalents by the light-driven ox-

idation of H

O (see below), photosynthetic bacteria must

obtain their reducing equivalents from the environment.

912 Chapter 24. Photosynthesis

JWCL281_c24_901-939.qxd 3/25/10 12:04 PM Page 912

Various substances, such as H

S, S, S

2⫺

, and many or-

ganic compounds, function in this capacity depending on

the bacterial species.

Modern photosynthetic bacteria are thought to resemble

the original photosynthetic organisms. These presumably

arose very early in the history of cellular life when environ-

mentally supplied sources of “high-energy” compounds

were dwindling but reducing agents were still plentiful (Sec-

tion 1-5Cb). During this era, photosynthetic bacteria were

no doubt the dominant form of life. However, their very

success eventually caused them to exhaust the available re-

ductive resources. The ancestors of modern cyanobacteria

adapted to this situation by evolving a photosynthetic sys-

tem with sufficient electromotive force to abstract electrons

from H

O. The gradual accumulation of the resulting toxic

waste product, O

, forced photosynthetic bacteria, which

cannot photosynthesize in the presence of O

(although

some species have evolved the ability to respire), into the

narrow ecological niches to which they are presently con-

fined (Section 1-1Ab).

C. Two-Center Electron Transport

See Guided Exploration 22: Two-center photosynthesis (Z-scheme)

overview

Plants and cyanobacteria use the reducing power

generated by the light-driven oxidation of H

O to produce

Section 24-2. Light Reactions 913

heme 4

heme 3

heme 2

heme 1

Four-heme c-type cytochrome

(not present in all species)

Exterior

(periplasm)

Cytoplasm

Plasma

membrane

hν

P870

Q cycle

BPheo a

Photosynthetic

reaction center

Cytochrome bc

cyt

cyt b

[2Fe–2S]

cyt c

cyt b

(a)

+0.6

+0.4

+0.2

Q pool

Cytochrome c

–0.2

–0.4

–0.6

BPheo b

–0.8

–1.0

P960*

hν

Cytochrome

complex

P960

′

(V)

(b)

Figure 24-13 Photosynthetic electron-transport system of

purple photosynthetic bacteria. (a) Schematic diagram indicating

the arrangement of the system components in the bacterial

plasma membrane and the flows of electrons (black arrows) and

protons (blue arrows) that photon (h␯) absorption promotes

through them.The system contains two protein complexes, the

RC and cytochrome bc

.Two electrons liberated from the special

pair, here P870 (as in Rb. sphaeroides), by the consecutive

absorption of two photons are taken up by ubiquinone (Q

)

together with two protons from the cytoplasm to yield ubiquinol

(QH

).The QH

is released from the RC and diffuses (dotted

arrows) through the membrane to cytochrome bc

, which, in a

two-electron reaction, oxidizes it to ubiquinone with the con-

comitant liberation of its two protons to the external medium.

One of the two electrons is passed, via the [2Fe–2S] cluster and

cytochrome c

, to cytochrome c

, a peripheral membrane protein

that then diffuses across the external surface of the membrane so

as to return the electron to P870 of the RC.The second electron

from QH

passes, via a Q cycle, through hemes b

and b

cytochrome bc

and then contributes to the reduction of a

molecule of ubiquinone (Q) with the concomitant uptake of two

more cytoplasmic protons (two rounds of a Q cycle are required

for the reduction of one molecule of Q to QH

; Fig. 22-31). The

resulting QH

diffuses back to cytochrome bc

.There it is again

oxidized, with the liberation of its two protons to the exterior

and the return of one of its two electrons, via cytochrome c

,to

P870, thereby completing the electrical circuit. Note that in every

turn of a Q cycle, half the electrons liberated by the oxidation of

to Q are used to reduce Q to QH

, so that, after a large

number of turns, an electron that enters the Q cycle, on average,

passes through it twice before being returned to P870.Thus, the

net result of the absorption of two photons by the RC is the

translocation of four H

⫹

from the cytoplasm to the external

medium. (b) The approximate standard reduction potentials of

the photosynthetic electron-transport system’s various

components.The overall process is essentially irreversible

because electrons are transferred to progressively lower energy

states (more positive standard reduction potentials).

JWCL281_c24_901-939.qxd 6/8/10 8:54 AM Page 913

0.08

quantum yield

0.10

0.12

0.14

0.06

0.04

0.02

660 680 700 720

Wavelength (nm)

Absence

of yellow-

green light

Presence

of yellow-

green light

NADPH. The component half-reactions of this process, to-

gether with their standard reduction potentials, are

and

Hence, the overall four-electron reaction and its standard

redox potential is

This latter quantity corresponds (Eq. [16.5]) to a standard

free energy change of ⌬G°¿ ⫽ 438 kJ ⴢ mol

⫺1

, which Eq.

[24.1] indicates is the energy of one einstein of 223-nm

photons (UV light). Clearly, even if photosynthesis were

100% efficient, which it is not, it would require more than

one photon of visible light to generate a molecule of O

. In

fact, experimental measurements indicate that algae mini-

mally require 8 to 10 photons of visible light to produce

one molecule of O

. In the following subsections, we dis-

cuss how plants and cyanobacteria manage this multipho-

ton process.

a. Photosynthetic O

Production Requires

Two Sequential Photosystems

Two seminal observations led to the elucidation of the

basic mechanism of photosynthesis in plants:

1. The quantum yield for O

evolution by Chlorella

pyrenoidosa varies little with the wavelength of the illumi-

nating light between 400 and 675 nm, but decreases precip-

itously above 680 nm (Fig. 24-14, lower curve). This phe-

nomenon, the “red drop,” was unexpected because Chl a

absorbs such far-red light (Fig. 24-5).

2. Shorter wavelength light, such as yellow-green

light, enhances the photosynthetic efficiency of 700-nm

light well in excess of the energy content of the shorter

wavelength light; that is, the rate of O

evolution by both

lights is greater than the sum of the rates for each light act-

ing alone (Fig. 24-14, upper curve). Moreover, this en-

hancement still occurs if the yellow-green light is

switched off several seconds before the red light is turned

on and vice versa.

These observations clearly indicate that two processes

are involved. They are explained by a mechanistic model,

the Z-scheme, which postulates that O

-producing photo-

synthesis occurs through the actions of two photosynthetic

RCs that are connected essentially in series (Fig. 24-15).

1. Photosystem I (PSI) generates a strong reductant

capable of reducing NADP

⫹

and, concomitantly, a weak

oxidant.

2. Photosystem II (PSII) generates a strong oxidant

capable of oxidizing H

O and, concomitantly, a weak re-

ductant.

e°¿ ⫽⫺1.135V

2NADP

⫹

⫹ 2H

O Δ 2NADPH ⫹ O

⫹ 2H

⫹

NADP

⫹

⫹ H

⫹

⫹ 2e

⫺

Δ NADPH

e°¿ ⫽⫺0.320V

⫹ 4e

⫺

⫹ 4H

⫹

Δ 2H

e°¿ ⫽⫹0.815V

The weak reductant reduces the weak oxidant, so that PSI

and PSII form a two-stage electron “energizer.” Both photo-

systems must therefore function for photosynthesis (electron

transfer from H

O to NADP

⫹

, forming O

and NADPH) to

occur.

The red drop is explained in terms of the Z-scheme by

the observation that PSII is only poorly activated by 680-

nm light. In the presence of only this far-red light, PSI is

activated but is unable to obtain more than a few of the

electrons it is capable of energizing. Yellow-green light,

however,efficiently stimulates PSII to supply these electrons.

The observation that the far-red and yellow-green lights

can be alternated indicates that both photosystems remain

activated for a time after the light is switched off.

The validity of the Z-scheme was established as follows.

The oxidation state of cytochrome f, a c-type cytochrome of

the electron-transport chain connecting PSI and PSII (see

below), can be spectroscopically monitored. Illumination

of algae with 680-nm (far-red) light results in the oxidation

of cytochrome f (Fig. 24-16). However, the additional

imposition of a 562-nm (yellow-green) light results in this

914 Chapter 24. Photosynthesis

Figure 24-14 Quantum yield for O

production by Chlorella

algae as a function of the wavelength of the incident light. The

experiment was conducted in the absence (lower curve) and the

presence (upper curve) of supplementary yellow-green light.The

upper curve has been corrected for the amount of O

production

stimulated by the supplementary light alone. Note that the lower

curve falls off precipitously above 680 nm (the red drop).

However, the supplementary light greatly increases the quantum

yield in the wavelength range above 680 nm (far-red) in which

the algae absorb light. [After Emerson, R., Chalmers, R., and

Cederstrand, C., Proc. Natl. Acad. Sci. 49, 137 (1957).]

JWCL281_c24_901-939.qxd 6/8/10 8:54 AM Page 914

protein’s partial re-reduction. In the presence of the herbi-

cide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU),

which abolishes photosynthetic oxygen production, 680-

nm light still oxidizes cytochrome f but simultaneous 562-

nm light only oxidizes it further.The explanation for these

effects is that 680-nm light, which efficiently activates only

PSI, causes it to withdraw electrons from (oxidize) cy-

tochrome f. The 562-nm light also activates PSII, which

thereby transfers electrons to (reduces) cytochrome f.

DCMU blocks electron flow from PSII to cytochrome f

(Fig. 24-15), so an increased intensity of light, whatever its

wavelength, only serves to activate PSI further.

b. O

-Producing Photosynthesis Is Mediated by

Three Transmembrane Protein Complexes

Linked by Mobile Electron Carriers

The components of the Z-scheme, which mediate electron

transport from H

O to NADPH, are largely organized into

three thylakoid membrane-bound particles (Fig. 24-17): (1)

3-(3,4-Dichlorophenyl)-1,1-dimethylurea

(DCMU)

PSII, (2) the cytochrome b

f complex, and (3) PSI. As in

oxidative phosphorylation, electrons are transferred be-

tween these complexes via mobile electron carriers. The

ubiquinone analog plastoquinone (Q), via its reduction to

plastoquinol (QH

CH C CH

Plastoquinone

2 [H•]

CH C

Plastoquinol

Section 24-2. Light Reactions 915

Figure 24-15 The Z-scheme for photosynthesis in plants and

cyanobacteria. Two photosystems, PSI and PSII, function to drive

electrons from H

O to NADPH. The reduction potential increases

downward so that electron flow occurs spontaneously in this

direction.The herbicide DCMU (see text) blocks photosynthetic

electron transport from PSII to cytochrome f.

Figure 24-16 The oxidation state of cytochrome f in

Porphyridium cruentum algae as monitored by a weak beam of

420-nm (blue-violet) light. An increase in the transmitted light

signals the oxidation of cytochrome f. In the upper curve, strong

light at 680 nm (far-red) causes the oxidation of the cytochrome f

but the superposition of 562-nm (yellow-green) light causes its

partial re-reduction. In the lower curve, the presence of the

herbicide DCMU, which inhibits photosynthetic electron

transport, causes 562-nm light to further oxidize, rather than

reduce, the cytochrome f.

Strong

oxidant

Increasing

reduction

potential

hν

–

Weak

reductant

+ 4H

cytochrome f

DCMU

Strong

reductant

NADP

+ H

NADPH

Weak

oxidant

hν

PSII

PSI

–

additions

Light transmitted at 420 nm

DCMU

added

On On Off Off

680 nm 562 nm 562 nm 680 nm

0 10203040

Time (s)

JWCL281_c24_901-939.qxd 3/25/10 12:04 PM Page 915

links PSII to the cytochrome b

f complex, which, in turn,

interacts with PSI through the mobile Cu-containing redox

protein plastocyanin (PC). In what follows, we trace the

electron pathway through this chloroplast system from

O to NADP

⫹

(Fig. 24-18).

c. PSII Resembles the PbRC

PSII from the thermophilic cyanobacterium Ther-

mosynechococcus elongatus consists of 20 subunits, 14 of

which occupy the photosynthetic membrane. These trans-

membrane subunits include the reaction center proteins

D1 (PsbA) and D2 (PsbD), the chlorophyll-containing

inner-antenna subunits CP43 (PsbC) and CP47 (PsbB), and

cytochrome b

559

.The X-ray structure of this PSII (Fig. 24-19),

independently determined by James Barber and So Iwata

and by Wolfram Saenger, reveals that this ⬃340-kD protein

is a symmetric dimer, whose protomeric units each contain

35 transmembrane helices. Each protomer, which has

pseudo-2-fold symmetry, binds 36 Chl a’s, 2 pheophytin a’s

(Pheo a’s; Chl a with its Mg

2⫹

replaced by two protons),

one heme b, one heme c, 2 plastoquinones, one nonheme

Fe, 12 all-trans carotenoids presumed to be ␤-carotene,

one ion, and one Mn

CaO

complex known as the

oxygen-evolving center [OEC; alternatively, the water-

oxidizing complex (WOC)]. In higher plants, the PSII pro-

tomer contains ⬃25 subunits and forms an ⬃1000-kD

transmembrane supercomplex with several antenna pro-

teins. The arrangement of the 5 transmembrane helices in

both D1 and D2 resembles that in the L and M subunits of

the PbRC (Fig. 24-11). Indeed, these two sets of subunits

have similar sequences, thereby indicating that they arose

from a common ancestor.

The cofactors of PSII’s RC (Fig. 24-20) are organized

similarly to those of the bacterial system (Fig. 24-12): They

have essentially the same components (with Chl a, Pheo a,

and plastoquinone replacing BChl b, BPheo b, and

menaquinone, respectively) and are symmetrically organ-

ized along the complex’s pseudo-2-fold axis. The two Chl a

rings labeled P

and P

in Fig. 24-20 are positioned anal-

ogously to the BChl b’s of P960’s “special pair” and are

therefore presumed to form PSII’s primary electron donor,

P680 (named after the wavelength at which its absorbance

HCO

⫺

916 Chapter 24. Photosynthesis

Figure 24-17 Schematic representation of the thylakoid

membrane showing the components of its electron-transport

chain. The system consists of three protein complexes: PSII, the

cytochrome b

f complex, and PSI, which are electrically

“connected” by the diffusion of the electron carriers plastoquinol

(Q) and plastocyanin (PC). Light-driven transport of electrons

(black arrows) from H

O to NADP

⫹

forming NADPH motivates

the transport of protons (red arrows) into the thylakoid space

(Fd is ferredoxin).Additional protons are split off from water by

Proton

translocating

ATP synthase

Fd–NADP

reductase

PSII

complex

Cytochrome b

PSI

complex

Outer membrane

Inner membrane

Thylakoid

Chloroplast

Stroma

+ O

hν

+ 2NADP

2NADPH

ADP + P

ATP

Stroma

Thylakoid

lumen

2QH

Cyt b

FeS

Cyt f

P700

P680

OEC

Pheo

cycle

FAD

FeS

hν

the oxygen-evolving complex (OEC), yielding O

.The resulting

proton gradient powers the synthesis of ATP by the CF

proton-translocating ATP synthase [CF

and CF

are chloroplast

and F

].The membrane also

contains light-harvesting complexes whose component

chlorophylls and other chromophores transfer their excitations

to PSI and PSII. [After Ort, D.R. and Good, N.E., Trends

Biochem. Sci. 13, 469 (1988).]

JWCL281_c24_901-939.qxd 6/8/10 8:54 AM Page 916

maximally decreases on photooxidation). The electron

ejected from P680 follows a similar asymmetric course as

that in the PbRC even though the two systems operate

over different ranges of reduction potential (compare Figs.

24-13b and 24-18). As indicated in the central part of Fig.

24-18, the electron is transferred to a molecule of Pheo a

(Pheo

in Fig. 24-20), probably via a Chl a molecule (Chl

and then to a bound plastoquinone (Q

).The electron is sub-

sequently transferred to a second plastoquinone molecule,

, which after it receives a second electron in a like manner,

takes up two protons at the stromal (cytosolic in cyanobacte-

ria) surface of the thylakoid membrane.The resulting plasto-

quinol, Q

, then exchanges with a membrane-bound pool

of plastoquinone molecules. DCMU as well as many other

commonly used herbicides compete with plastoquinone for

the Q

-binding site on PSII, which explains how they inhibit

photosynthesis.

Two “extra” Chl a molecules, Chlz

and Chlz

, lie on

the periphery of the RC,where they are postulated to func-

tion in the transfer of excitation from the antenna systems

to P680. Cytochrome b

559

, whose function is unclear, breaks

the pseudosymmetry of the PSII protomer as does the Mn

cluster, whose function we now discuss.

d. O

Is Generated in a Five-Stage Water-Splitting

Reaction Mediated by an Mn-Containing

Protein Complex

The oxidation by the OEC of two molecules of H

O to

form one molecule of O

requires four electrons. Since

transfer of a single electron from H

O to NADP

⫹

requires

two photochemical events, this accounts for the observed

minimum of 8 to 10 photons absorbed per molecule of O

produced.

Must the four electrons necessary to produce a given O

molecule be removed by a single photosystem or can they

be extracted by several different photosystems? Pierre

Joliet and Bessel Kok answered this question by analyzing

the rate at which dark-adapted chloroplasts produce O

Section 24-2. Light Reactions 917

Figure 24-18 Detailed diagram of the Z-scheme of

photosynthesis. Electrons ejected from P680 by the absorption of

photons are replaced with electrons abstracted from H

O by an

Mn complex (OEC), thereby forming O

and four H

⫹

. Each

ejected electron is passed through a chain of electron carriers

to a pool of plastoquinone molecules (Q).The resulting

plastoquinol, in turn, reduces the cytochrome b

f particle (yellow

box) that transfers electrons with the concomitant translocation

of protons, via a Q cycle, into the thylakoid lumen. Cytochrome

•

hν

+ 4H

–

(thylakoid)

complex

.....

P680

cyt b

[4Fe–4S]

cyt f

Chl a

Pheo a

pool

Cytochrome

Cyclic

pathway

(stroma)

(thylakoid)

P700

noncyclic

pathway

–

2NADP

FNR

(stroma)

FQR

2NADPH

PSI

PSII

–1.0

–0.8

–1.2

–1.4

–0.6

–0.4

–0.2

•

OEC

hν

+0.6

+0.8

+1.0

+1.2

+0.4

+0.2

′

(V)

f then transfers the electrons to plastocyanin (PC).The

plastocyanin regenerates photooxidized P700.The electron

ejected from P700, through the intermediacy of a chain of

electron carriers (A

, and Fd), reduces NADP

⫹

NADPH in noncyclic electron transport.Alternatively, the

electron may be returned to the cytochrome b

f complex in a

cyclic process that only translocates protons into the thylakoid

lumen.

JWCL281_c24_901-939.qxd 3/25/10 12:04 PM Page 917

when exposed to a series of short flashes. O

was evolved

with a peculiar oscillatory pattern (Fig. 24-21). There is vir-

tually no O

evolved by the first two flashes.The third flash

results in the maximal O

yield. Thereafter, the amount of

produced peaks with every fourth flash until the oscilla-

tions damp out to a steady state. This periodicity indicates

that each OEC cycles through five different states, S

through S

(Fig. 24-22). Each of the transitions between S

and S

is a photon-driven redox reaction; that from S

to S

results in the release of O

.Thus, each O

molecule must be

produced by a single photosystem. The observation that O

evolution peaks at the third rather than the fourth flash in-

dicates that the OEC’s resting state is predominantly S

rather than S

.The oscillations gradually damp out because

a small fraction of the RCs fail to be excited or become

doubly excited by a given flash of light, so that they eventu-

ally lose synchrony.The five reaction steps release a total of

four water-derived protons into the inner thylakoid space

918 Chapter 24. Photosynthesis

Figure 24-19 X-ray structure of PSII from T. elongatus.

(a) The PSII dimer is viewed from within the plane of the

membrane with the stroma above. Its transmembrane subunits

include D1 (yellow), D2 (orange), CP47 (red), CP43 (green), and

cytochrome b

559

(magenta). Other transmembrane subunits are

colored light blue and blue-gray. Its membrane-extrinsic proteins

are PsbO (dark blue), PsbU (purple), and PsbV (light green). The

various cofactors are drawn in stick form with the chlorophylls of

the D1/D2 reaction center light green, those of the antenna

complexes dark green, pheophytins dark blue, hemes red,

␤-carotenes orange, Q

and Q

purple, and the nonheme Fe

represented by a red sphere. The inferred position of the

membrane is indicated by the light blue band. (b) View of a PSII

protomer perpendicular to the membrane from the thylakoid

lumen showing only the transmembrane portions of the complex

and colored as in Part a.A portion of the other protomer in the

PSII dimer is shown in muted colors with the dashed line

indicating the region of monomer–monomer interactions and the

black ellipse indicating the position of the 2-fold axis.The

pseudo-2-fold axis, which is perpendicular to the membrane and

passes through the nonheme Fe, relates the transmembrane

helices of the D1/D2 heterodimer, CP43 and CP47, and PsbI and

PsbX as emphasized by the black lines encircling these subunits.

[Courtesy of James Barber and So Iwata, Imperial College

London, U.K. PDBid 1S5L.]

(a)

(b)

JWCL281_c24_901-939.qxd 6/8/10 8:54 AM Page 918

(lumen) in a stepwise manner (Fig. 24-22). These protons

contribute to the transmembrane proton gradient.

Since the OEC abstracts electrons from H

O, its five

states must have extraordinarily high reduction potentials

(recall from Table 22-1 that the O

O half-reaction has a

standard reduction potential of 0.815 V). PSII must also

stabilize the highly reactive intermediates for extended pe-

riods (as much as minutes) in close proximity to water.

The OEC, which is located at the lumenal surface of the

D1 subunit (Fig. 24-20), is a Mn

CaO

or Mn

CaO

complex

in which the O atoms bridge neighboring Mn atoms. The

structure of the OEC remains elusive due to PSII’s rela-

tively poorly resolved X-ray structures and the observation

Section 24-2. Light Reactions 919

4 8 12 16 20 24

yield per flash

Flash number

–

hν

Figure 24-20 The arrangement of electron-transfer cofactors

in PSII from T. elongatus. The complex is viewed along the

membrane plane with the thylakoid lumen below. The cofactors

are colored as in Fig. 24-19 but with Mg

2⫹

yellow, N blue, and O

red.The phytyl tails of the chlorophylls and pheophytins have

been removed for clarity. The side chain C atoms of Tyr

(D1 Tyr

161) and D1 His 190 are yellow, and those of Tyr

(D2 Tyr 160)

Figure 24-21 The O

yield per flash in dark-adapted spinach

chloroplasts. Note that the yield peaks on the third flash and

then on every fourth flash thereafter until the curve eventually

damps out to its average value. [After Forbush, B., Kok, B., and

McGloin, M.P., Photochem. Photobiol. 14, 309 (1971).]

Figure 24-22 Schematic mechanism of O

generation in

chloroplasts. Four electrons are stripped, one at a time in light-

driven reactions (S

S S

), from two bound H

O molecules. In

the recovery step (S

S S

), which is light independent, O

released and two more H

O molecules are bound.Three of these

five steps release protons into the thylakoid lumen.

and D2 His 189 are orange. The OEC is drawn in space-filling form

with Mn purple, Ca

2⫹

cyan, and O red.The numbers indicate the

center-to-center distances, in angstroms, between the cofactors

spanned by the accompanying thin black lines. Compare this

figure to Fig. 24-12 (which is drawn upside down relative to this

figure). [Courtesy of James Barber and So Iwata, Imperial

College London, U.K. PDBid 1S5L.]

JWCL281_c24_901-939.qxd 6/16/10 12:04 PM Page 919

that the OEC decomposes when illuminated with X-rays at

the intensities used in X-ray structure determinations. How-

ever, the use of X-ray spectroscopy techniques of lower in-

tensity that are sensitive to bond lengths have led to the

formulation of several related models for the OEC that

are compatible with the X-ray structure of PSII. One of

these models is shown in Fig. 24-23.

The water-splitting reaction is driven by the excitation

of the PSII RC.A variety of evidence indicates that the Mn

ions in the OEC’s various S states (Fig. 24-22) cycle

through specific combinations of Mn(II), Mn(III), Mn(IV),

and Mn(V) while abstracting protons and electrons from

two H

O molecules to yield O

, which is released into the

thylakoid lumen. However, the mechanism whereby this

occurs, that is, the nature of the five S states, remains un-

known due to the lack of structural information concerning

these states.

The next link in the PSII electron transport chain is an

entity, originally named Z (Fig. 24-18), which relays elec-

trons from the OEC to P680.The existence of Z is signaled

by a transient EPR spectrum of illuminated chloroplasts

that parallels the S-state transitions. The change in this

spectrum on supplying deuterated tyrosine to cyanobacte-

ria indicates that Z

⫹

is a tyrosyl radical (TyrOⴢ; EPR spec-

tra reflect the nuclear spins of the atoms with which the un-

paired electrons interact). It has been identified as Tyr

PSII (Fig. 24-20) due to its position between the Mn cluster

and P680’s chlorophyll P

. Recall that a tyrosyl radical has

also been implicated in the reduction of O

to 2 H

O by

cytochrome c oxidase (Complex IV) in the respiratory

electron-transport chain (Section 22-2C5c).

e. Electron Transport through the Cytochrome

f Complex Generates a Proton Gradient

From the plastoquinone pool, electrons pass through

the cytochrome b

f complex. This integral membrane as-

sembly resembles cytochrome bc

, its purple bacterial

counterpart (Section 24-2Be), as well as Complex III of the

mitochondrial electron-transport chain (also called cy-

tochrome bc

; Section 22-2C3a). Electron flow through the

cytochrome b

f complex occurs through a Q cycle (Fig.

22-31) in which plastoquinone is the (H

⫹

⫹ e

⫺

) carrier.

Accordingly, two protons are translocated across the thy-

lakoid membrane for every electron transported. The four

electrons abstracted from 2 H

O by the OEC therefore

lead to the translocation of eight H

⫹

from the stroma to the

thylakoid lumen. Electron transport via the cytochrome b

complex generates much of the electrochemical proton gra-

dient that drives the synthesis of ATP in chloroplasts.

The X-ray structure of cytochrome b

f (Fig. 24-24) was

independently determined by Janet Smith and William

Cramer and by Jean-Luc Popot and Daniel Picot. Cy-

tochrome b

f is a dimer of ⬃109-kD protomers, each con-

taining four large subunits (18–32 kD) that have counter-

parts in cytochrome bc

: cytochrome b

, a homolog of the

N-terminal half of cytochrome b; subunit IV, a homolog of

the C-terminal half of cytochrome b; a Rieske iron–sulfur

protein (ISP), which is also present in cytochrome bc

; and

cytochrome f (f for feuille, French for leaf), a c-type cy-

tochrome that is a functional analog of cytochrome c

,al-

though the two are unrelated in structure or sequence. In

fact, cytochrome f is an elongated, two-domain protein that

is dominated by ␤ sheets and hence has an entirely differ-

ent fold from those of other c-type cytochromes of known

structure. Cytochrome f’s single heme c is, nevertheless, co-

valently linked to the protein’s larger domain via the two

Cys residues in a Cys-X-Y-Cys-His sequence that is charac-

teristic of c-type cytochromes (Fig. 9-41) and whose His

residue forms one of the Fe ion’s two axial ligands (Fig.22-21).

Intriguingly, however, the second axial ligand is not a Met

S atom, as occurs in most c-type cytochromes, but instead is

the protein’s N-terminal amino group, a group that had

previously not been observed to be a heme ligand.

In addition, cytochrome b

f has four small hydrophobic

subunits that have no equivalents in cytochrome bc

. Each

protomer contains 13 transmembrane helices, four in cy-

tochrome b

, three in subunit IV, and one each in the re-

maining subunits. Cytochrome b

f binds cofactors that are

the equivalents of all of those in cytochrome bc

: heme f, a

c-type heme bound by cytochrome f; a [2Fe–2S] cluster

bound by the ISP; hemes b

and b

; a plastoquinone mole-

cule that occupies either the Q

site (the quinone-binding

site at which fully reduced quinone is regenerated during

the Q cycle; Section 22-3Be) or the Q

site. In addition,

cytochrome b

f binds several cofactors that have no coun-

terparts in cytochrome bc

: a Chl a, a ␤-carotene, and, un-

expectedly, a novel heme named heme x (alternatively,

heme c

), which is covalently linked to the protein via a sin-

gle thioether bond to Cys 35 of cytochrome b

, and whose

only axial ligand is a water molecule (compare with hemes

a, b, and c; Fig. 22-21).

920 Chapter 24. Photosynthesis

Figure 24-23 A model of the OEC. This Mn

CaO

complex is

shown in ball-and-stick form with Mn ions purple, the Ca

2⫹

ion

cyan, and O red.The bonds between the Ca and Mn ions are

drawn in gray to indicate that the position of the Ca ion is rela-

tively poorly defined. Presumably, numerous protein side chains

and water molecules ligand the Ca and Mn ions. Several related

models are also compatible with the structural data. [Based on a

model by Vittal Yachandra, Lawrence Berkeley National

Laboratory, Berkeley, California.]

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