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

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resonance energy transfer. In blue-green and red algae, phycobilisomes large protein assemblies with bound pigments

called bilins act as light-absorbing antennas.

The Ability to Convert Light into Chemical Energy Is Ancient

The photosystems have structural features in common that suggest a common evolutionary origin. Similarities in

organization and molecular structure to those of oxidative phosphorylation suggest that the photosynthetic apparatus

evolved from an early energy-transduction system.

Key Terms

light reaction

chloroplast

stroma

thylakoid

granum

chlorophyll a

photoinduced charge separation

reaction center

photosystem I (PS I)

photosystem II (PS II)

special pair

P960

P680

manganese center

cytochrome bf

P700

Z scheme of photosynthesis

proton-motive force ( ∆p)

ATP synthase (CF

-CF

complex)

cyclic photophosphorylation

carotenoid

light-harvesting complex

phycobilisome

II. Transducing and Storing Energy 19. The Light Reactions of Photosynthesis

Problems

Electron transfer. Calculate the ∆ E

and ∆ G° for the reduction of NADP

by ferredoxin. Use data given in Table

18.1 in Section 18.2.1.

See answer

To boldly go. (a) It can be argued that, if life were to exist elsewhere in the universe, it would require some process

like photosynthesis. Why is this a reasonable argument? (b) If the Enterprise were to land on a distant plant and find

no measurable oxygen in the atmosphere, could the crew conclude that photosynthesis is not taking place?

See answer

Weed killer 1. Dichlorophenyldimethylurea (DCMU), a herbicide, interferes with photophosphorylation and O

evolution. However, it does not block O

evolution in the presence of an artificial electron acceptor such as

ferricyanide. Propose a site for the inhibitory action of DCMU.

See answer

Weed killer 2. Predict the effect of the herbicide dichlorophenyldimethylurea (DCMU) on a plant's ability to perform

cyclic photophosphorylation.

See answer

Infrared harvest. Consider the relation between the energy of a photon and its wavelength.

(a) Some bacteria are able to harvest 1000-nm light. What is the energy (in kilocalories or kilojoules) of a mole (also

called an einstein) of 1000-nm photons?

(b) What is the maximum increase in redox potential that can be induced by a 1000-nm photon?

? Assume a ∆ G of 12

kcal mol

-1

(50 kJ mol

-1

) for the phosphorylation reaction.

See answer

Missing acceptors. Suppose that a bacterial reaction center containing only the special pair and the quinones was

prepared. Given the separation of 22 Å between the special pair and the closest quinone, estimate the rate of electron

transfer between the excited special pair and this quinone.

See answer

Self-preservation. Blue-green bacteria deprived of a source of nitrogen digest their least-essential proteins. Which

phycobilisome component is likely to be degraded first under starvation conditions?

See answer

Close approach. Suppose that energy transfer between two chlorophyll a molecules separated by 10 Å occurs in 10

picoseconds. Suppose that this distance is increased to 20 Å with all other factors remaining the same. How long

would energy transfer take?

See answer

Mechanism Problem

Hill reaction. In 1939, Robert Hill discovered that chloroplasts evolve O

when they are illuminated in the presence

of an artificial electron acceptor such as ferricyanide [Fe

(CN)

]

. Ferricyanide is reduced to ferrocyanide [Fe

(CN)

]

in this process. No NADPH or reduced plastocyanin is produced. Propose a mechanism for the Hill

reaction.

See answer

Data Interpretation and Chapter Integration Problem

10.

The same, but different. The α

γ complex of mitochondrial or chloroplast ATP synthase will function as an

ATPase in vitro. The chloroplast enzyme (both synthase and ATPase activity) is sensitive to redox control, whereas

the mitochondrial enzyme is not. To determine where the enzymes differ, a segment of the mitochondrial γ subunit

was removed and replaced with the equivalent segment from the chloroplast γ subunit. The ATPase activity of the

modified enzyme was then measured as a function of redox conditions.

(a) What is the redox regulator of the ATP synthase in vivo?

The graph below shows the ATPase activity of modified and control enzymes under various redox conditions.

[Data after O. Bald, et al., 2000. J Biol. Chem., 275: 12757

12762.]

(b) What is the effect of increasing the reducing power of the reaction mixture for the control and modified

enzymes?

DTT alone? Suggest a possible explanation for the difference.

(d) Did the researchers succeed in identifying the region of the γ subunit responsible for redox regulation?

(e) What is the biological rationale of regulation by high concentrations of reducing agents?

(f) What amino acids in the γ subunit are most likely affected by the reducing conditions?

(g) What experiments might confirm your answer to part e?

See answer

II. Transducing and Storing Energy 19. The Light Reactions of Photosynthesis

Selected Readings

Where to start

R. Huber. 1989. A structural basis of light energy and electron transfer in biology EMBO J. 8: 2125-2147. (PubMed)

J. Deisenhofer and H. Michel. 1989. The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas

viridis EMBO J. 8: 2149-2170. (PubMed)

J. Barber and B. Andersson. 1994. Revealing the blueprint of photosynthesis Nature 370: 31-34.

Books and general reviews

Raghavendra, A. S., 1998. Photosynthesis: A Comprehensive Treatise . 1998. Cambridge University Press.

Cramer, W. A., and Knaff, D. B., 1991. Energy Transduction in Biological Membranes: A Textbook of Bioenergetics .

Springer Verlag.

Nicholls, D. G., and Ferguson, S. J., 1997. Bioenergetics (2d ed.). Academic Press.

Harold, F. M., 1986. The Vital Force: A Study of Bioenergetics . W. H. Freeman and Company.

Electron-transfer mechanisms

D. Beratan and S. Skourtis. 1998. Electron transfer mechanisms Curr. Opin. Chem. Biol. 2: 235-243. (PubMed)

C.C. Moser, J.M. Keske, K. Warncke, R.S. Farid, and P.L. Dutton. 1992. Nature of biological electron transfer Nature

355: 796-802. (PubMed)

S.G. Boxer. 1990. Mechanisms of long-distance electron transfer in proteins: Lessons from photosynthetic reaction

centers Annu. Rev. Biophys. Biophys. Chem. 19: 267-299. (PubMed)

Photosystem II

A. Zouni, H.T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, and P. Orth. 2001. Crystal structure of photosystem II

from Syne-chococcus elongatus at 3.8 Å resolution Nature 409: 739-743. (PubMed)

K.H. Rhee, E.P. Morris, J. Barber, and W. Kuhlbrandt. 1998. Three-dimensional structure of the plant photosystem II

reaction centre at 8 Å resolution Nature 396: 283-286. (PubMed)

E.P. Morris, B. Hankamer, D. Zheleva, G. Friso, and J. Barber. 1997. The three-dimensional structure of a photosystem

II core complex determined by electron crystallography Structure 5: 837-849. (PubMed)

J. Deisenhofer and H. Michel. 1991. High-resolution structures of photosynthetic reaction centers Annu. Rev. Biophys.

Biophys. Chem. 20: 247-266. (PubMed)

W. Vermaas. 1993. Molecular-biological approaches to analyze photosystem II structure and function Annu. Rev. Plant

Physiol. Plant Mol. Biol. 44: 457-481.

Oxygen evolution

C.W. Hoganson and G.T. Babcock. 1997. A metalloradical mechanism for the generation of oxygen from water in

photosynthesis Science 277: 1953-1956. (PubMed)

V.K. Yamachandra, V.J. DeRose, M.J. Latimer, I. Mukerji, K. Sauer, and M.P. Klein. 1993. Where plants make oxygen:

A structural model for the photosynthetic oxygen-evolving manganese complex Science 260: 675-679. (PubMed)

G.W. Brudvig, W.F. Beck, and J.C. de Paula. 1989. Mechanism of photosynthetic water oxidation Annu. Rev. Biophys.

Biophys. Chem. 18: 25-46. (PubMed)

J.M. Peloquin and R.D. Britt. 2001. EPR/ENDOR characterization of the physical and electronic structure of the OEC

Mn cluster Biochim. Biophys. Acta 1503: 96-111. (PubMed)

Photosystem I and cytochrome bf

W.D. Schubert, O. Klukas, W. Saenger, H.T. Witt, P. Fromme, and N. Krauss. 1998. A common ancestor for oxygenic

and anoxygenic photosynthetic systems: A comparison based on the structural model of photosystem I J. Mol. Biol. 280:

297-314. (PubMed)

D. Fotiadis, D.J. Muller, G. Tsiotis, L. Hasler, P. Tittmann, T. Mini, P. Jeno, H. Gross, and A. Engel. 1998. Surface

analysis of the photosystem I complex by electron and atomic force microscopy J. Mol. Biol. 283: 83-94. (PubMed)

O. Klukas, W.D. Schubert, P. Jordan, N. Krauss, P. Fromme, H.T. Witt, and W. Saenger. 1999. Photosystem I, an

improved model of the stromal subunits PsaC, PsaD, and PsaE J. Biol. Chem. 274: 7351-7360. (PubMed)

P.E. Jensen, M. Gilpin, J. Knoetzel, and H.V. Scheller. 2000. The PSI-K subunit of photosystem I is involved in the

interaction between light-harvesting complex I and the photosystem I reaction center core J. Biol. Chem. 275: 24701-

24708. (PubMed)

A. Kitmitto, A.O. Mustafa, A. Holzenburg, and R.C. Ford. 1998. Three-dimensional structure of higher plant

photosystem I determined by electron crystallography J. Biol. Chem. 273: 29592- 29599. (PubMed)

N. Krauss, W. Hinrichs, I. Witt, P. Fromme, W. Pritzkow, Z. Dauter, C. Betzel, K.S. Wilson, H.T. Witt, and W. Saenger.

1993. Three-dimensional structure of system I photosynthesis at 6 Å resolution Nature 361: 326-331. (PubMed)

R. Malkin. 1992. Cytochrome bc

and b

f complexes of photosynthetic membranes Photosynth. Res. 33: 121-136.

(PubMed)

P.A. Karplus, M.J. Daniels, and J.R. Herriott. 1991. Atomic structure of ferredoxin-NADP

reductase: Prototype for a

structurally novel flavoenzyme family Science 251: 60-66. (PubMed)

ATP synthase

M.L. Richter, R. Hein, and B. Huchzermeyer. 2000. Important subunit interactions in the chloroplast ATP synthase

Biochim. Biophys. Acta 1458: 326-329. (PubMed)

G. Oster and H. Wang. 1999. ATP synthase: Two motors, two fuels Structure 7: R67-R72. (PubMed)

W. Junge, H. Lill, and S. Engelbrecht. 1997. ATP synthase: An electrochemical transducer with rotatory mechanics

Trends Biochem. Sci. 22: 420-423. (PubMed)

J. Weber and A.E. Senior. 2000. ATP synthase: What we know about ATP hydrolysis and what we do not know about

ATP synthesis Biochim. Biophys. Acta 1458: 300-309. (PubMed)

Light-harvesting assemblies

M.J. Conroy, W.H. Westerhuis, P.S. Parkes-Loach, P.A. Loach, C.N. Hunter, and M.P. Williamson. 2000. The solution

structure of Rhodobacter sphaeroides LH1beta reveals two helical domains separated by a more flexible region:

Structural consequences for the LH1 complex J. Mol. Biol. 298: 83-94. (PubMed)

J. Koepke, X. Hu, C. Muenke, K. Schulten, and H. Michel. 1996. The crystal structure of the light-harvesting complex II

(B800 850) from Rhodospirillum molischianum Structure 4: 581-597. (PubMed)

A.R. Grossman, D. Bhaya, K.E. Apt, and D.M. Kehoe. 1995. Light-harvesting complexes in oxygenic photosynthesis:

Diversity, control, and evolution Annu. Rev. Genet. 29: 231-288. (PubMed)

W. Kühlbrandt, D.-N. Wang, and Y. Fujiyoshi. 1994. Atomic model of plant light-harvesting complex by electron

crystallography Nature 367: 614-621. (PubMed)

A.N. Glazer. 1983. Comparative biochemistry of photosynthetic light-harvesting systems Annu. Rev. Biochem. 52: 125-

157. (PubMed)

Evolution

B.R. Green. 2001. Was "molecular opportunism" a factor in the evolution of different light-harvesting photosynthetic

light-harvesting pigment systems? Proc. Natl. Acad. Sci. USA 98: 2119-2121. (PubMed) (Full Text in PMC)

G.C. Dismukes, V.V. Klimov, S.V. Baranov, Y.N. Nozlov, J. DasGupta, and A. Tyryshkin. 2001. The origin of

atmospheric oxygen on earth: The innovation of oxygenic photosynthesis Proc. Natl. Acad. Sci. USA 98: 2170-2175.

(PubMed) (Full Text in PMC)

D. Moreira, H. Le Guyader, and H. Phillippe. 2000. The origin of red algae and the evolution of chloroplasts Nature 405:

69-72. (PubMed)

T. Cavalier-Smith. 2000. Membrane heredity and early chloroplast evolution Trends Plant Sci. 5: 174-182. (PubMed)

R.E. Blankenship and H. Hartman. 1998. The origin and evolution of oxygenic photosynthesis Trends Biochem. Sci. 23:

94-97. (PubMed)

II. Transducing and Storing Energy

20. The Calvin Cycle and the Pentose Phosphate Pathway

Photosynthesis proceeds in two parts: the light reactions and the dark reactions. The light reactions, which were

discussed in Chapter 19, transform light energy into ATP and biosynthetic reducing power, NADPH. The dark reactions,

which constitute the Calvin cycle, named after Melvin Calvin, the biochemist who elucidated the pathway, reduce carbon

atoms from their fully oxidized state as carbon dioxide to the more reduced state as a hexose. The components of the

Calvin cycle and called the dark reactions because, in contrast with the light reactions, these reactions do not directly

depend on the presence of light.

In addition to ATP, the dark reactions require reducing power in the form of NADPH, the currency of readily available

reducing power in cells. The phosphoryl group on the 2

-hydroxyl group of one of the ribose units of NADPH

distinguishes NADPH from NADH. There is a fundamental distinction between NADPH and NADH in biochemistry:

NADH is oxidized by the respiratory chain to generate ATP, whereas NADPH serves as a reductant in biosynthetic

processes.

The second half of this chapter examines a pathway common to all organisms, known variously as the pentose phosphate

pathway, the hexose monophosphate pathway, the phosphogluconate pathway, or the pentose shunt. The pathway

provides a means by which glucose can be oxidized to generate NADPH and is the source of much of the NADPH that is

needed for the biosynthesis of many biomolecules, most notably fats. We will observe the use of NADPH in many of the

biosynthetic reactions considered in Part III of this text. The pentose phosphate pathway can also be used for the

catabolism of pentose sugars from the diet, the synthesis of pentose sugars for nucleotide biosynthesis, and the

catabolism and synthesis of less common four- and seven-carbon sugars. The pentose phosphate pathway and the Calvin

cycle have in common several enzymes and intermediates that attest to an evolutionary kinship. Like glycolysis and

gluconeogenesis (Chapter 16), these pathways are mirror images of one another: the Calvin cycle uses NADPH to reduce

carbon dioxide to generate hexoses, whereas the pentose phosphate pathway breaks down glucose into carbon dioxide to

generate NADPH. The Calvin cycle is sometimes referred to as the reductive pentose phosphate pathway.

II. Transducing and Storing Energy 20. The Calvin Cycle and the Pentose Phosphate Pathway

Atmospheric carbon dioxide measurements as Mauna Loa, Hawaii. These measurements show annual cycles

resulting from seasonal variation in carbon dioxide fixation by the Calvin cycle in terrestrial plants. Much of this fixation

takes place in rain forests, which account for approximately 50% of terrestrial fixation. [Dennis Potokar/Photo

Researchers.]

II. Transducing and Storing Energy 20. The Calvin Cycle and the Pentose Phosphate Pathway

20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water

We saw in Chapter 16 that glucose could be formed from noncarbohydrate precursors, such as lactate and amino acids,

by gluconeogenesis. The synthesis of glucose from these compounds is simplified because the carbons are already

incorporated into relatively complex organic molecules. In contrast, the source of the carbon atoms in the Calvin cycle is

the simple molecule carbon dioxide. In this extremely important process, carbon dioxide gas is trapped in a form that is

useful for many processes. The Calvin cycle brings into living systems the carbon atoms that will become constituents of

nucleic acids, proteins, and fats. Photosynthetic organisms are called autotrophs (literally "self-feeders") because they

can synthesize glucose from carbon dioxide and water, by using sunlight as an energy source, and then recover some of

this energy from the synthesized glucose through the glycolytic pathway and aerobic metabolism. Organisms that obtain

energy from chemical fuels only are called heterotrophs, which ultimately depend on autotrophs for their fuel. The

Calvin cycle also differs from gluconeogenesis in where it takes place in photosynthetic eukaryotes. Whereas

gluconeogenesis takes place in the cytoplasm, the Calvin cycle takes place in the stroma of chloroplasts, the

photosynthetic organelles.

The Calvin cycle comprises three stages (Figure 20.1):

1. The fixation of CO

by ribulose 1,5-bisphosphate to form two molecules of 3-phosphoglycerate.

2. The reduction of 3-phosphoglycerate to form hexose sugars.

3. The regeneration of ribulose 1,5-bisphosphate so that more CO

can be fixed.

Although we will focus on the Calvin cycle, other means of fixing carbon dioxide into hexose sugars exist in the

photosynthetic world, notably a version of the citric acid cycle running in reverse.

20.1.1. Carbon Dioxide Reacts with Ribulose 1,5-bisphosphate to Form Two Molecules

of 3-Phosphoglycerate

The first step in the Calvin cycle is the fixation of CO

. The CO

molecule condenses with ribulose 1,5-bisphosphate to

form an unstable six-carbon compound, which is rapidly hydrolyzed to two molecules of 3-phosphoglycerate.

The initial incorporation of CO

into 3-phosphoglycerate was revealed through the use of a carbon-14 radioactive tracer

(Figure 20.2). This highly exergonic reaction [∆ G°´ = 12.4 kcal mol

( 51.9 kJ mol

)] is catalyzed by ribulose 1,5-

bisphosphate carboxylase/oxygenase (usually called rubisco), an enzyme located on the stromal surface of the thylakoid

membranes of chloroplasts. This important reaction is the rate-limiting step in hexose synthesis. Rubisco in chloroplasts

consists of eight large (L, 55-kd) subunits and eight small (S, 13-kd) ones (Figure 20.3). Each L chain contains a

catalytic site and a regulatory site. The S chains enhance the catalytic activity of the L chains. This enzyme is very

abundant in chloroplasts, constituting more than 16% of their total protein. In fact, rubisco is the most abundant enzyme

and probably the most abundant protein in the biosphere. Large amounts are present because rubisco is a slow enzyme;

its maximal catalytic rate is only 3 s

Rubisco requires a bound divalent metal ion for activity, usually magnesium ion. Like the zinc ion in the active site of

carbonic anhydrase (Section 9.2.1), this metal ion serves to activate a bound substrate molecule by stabilizing a negative

charge. Interestingly, a CO

molecule other than the substrate is required to complete the assembly of the Mg

binding

site in rubisco. This CO

molecule adds to the uncharged ε-amino group of lysine 201 to form a carbamate. This

negatively charged adduct then binds the Mg

ion. The formation of the carbamate is facilitated by the enzyme rubisco

activase, although it will also form spontaneously at a lower rate.

The metal center plays a key role in binding ribulose 1,5-bisphosphate and activating it so that it will react with CO

(Figure 20.4). Ribulose 1,5-bisphosphate binds to Mg

through its keto group and an adjacent hydroxyl group. This

complex is readily deprotonated to form an enediolate intermediate. This reactive species, analogous to the zinc-

hydroxide species in carbonic anhydrase (Section 9.2.2), couples with CO

, forming the new carbon-carbon bond. The

resulting product is coordinated to the Mg

ion through three groups, including the newly formed carboxylate. A

molecule of H

O is then added to this β-ketoacid to form an intermediate that cleaves to form two molecules of 3-

phosphoglycerate (Figure 20.5).

20.1.2. Catalytic Imperfection: Rubisco Also Catalyzes a Wasteful Oxygenase Reaction

The reactive intermediate generated on the Mg

ion sometimes reacts with O

instead of CO

. Thus, rubisco also

catalyzes a deleterious oxygenase reaction. The products of this reaction are phosphoglycolate and 3-phosphoglycerate

(Figure 20.6). The rate of the carboxylase reaction is four times that of the oxygenase reaction under normal atmospheric

conditions at 25°C; the stromal concentration of CO

is then 10 µM and that of O

is 250 µM. The oxygenase reaction,

like the carboxylase reaction, requires that lysine 201 be in the carbamate form. Because this carbamate forms only in the

presence of CO

, this property would prevent rubisco from catalyzing the oxygenase reaction exclusively when CO

absent.

Phosphoglycolate is not a versatile metabolite. A salvage pathway recovers part of its carbon skeleton (Figure 20.7). A

specific phosphatase converts phosphoglycolate into glycolate, which enters peroxisomes (also called microbodies;

Figure 20.8). Glycolate is then oxidized to glyoxylate by glycolate oxidase, an enzyme with a flavin mononucleotide

prosthetic group. The H

produced in this reaction is cleaved by catalase to H

O and O

. Transamination of

glyoxylate then yields glycine. Two glycine molecules can be used to form serine, a potential precursor of glucose, with

the release of CO

and ammonia (NH

). The ammonia, used in the synthesis of nitrogen-containing compounds, is

salvaged by glutamine synthetase reaction.

This salvage pathway serves to recycle three of the four carbon atoms of two molecules of glycolate. However, one

carbon atom is lost as CO

. This process is called photorespiration because O

is consumed and CO

is released.

Photorespiration is wasteful because organic carbon is converted into CO

without the production of ATP, NADPH, or

another energy-rich metabolite. Moreover, the oxygenase activity increases more rapidly with temperature than the

carboxylase activity, presenting a problem for trop-ical plants (Section 20.2.3). Evolutionary processes have presumably

enhanced the preference of rubisco for carboxylation. For instance, the rubisco of higher plants is eightfold as specific

for carboxylation as that of photosynthetic bacteria.

20.1.3. Hexose Phosphates Are Made from Phosphoglycerate, and Ribulose 1,5-

bisphosphate Is Regenerated

The 3-phosphoglycerate product of rubisco is next converted into three forms of hexose phosphate: glucose 1-phosphate,

glucose 6-phosphate, and fructose 6-phosphate. Recall that these isomers are readily interconvertible (Sections 16.1.2

and 16.1.11). The steps in this conversion (Figure 20.9) are like those of the gluconeogenic pathway (Section 16.3.1),

except that glyceraldehyde 3-phosphate dehydrogenase in chloroplasts, which generates glyceraldehyde 3-phosphate

(GAP), is specific for NADPH rather than NADH. Alternatively, the glyceraldehyde 3-phosphate can be transported to

the cytosol for glucose synthesis. These reactions and that catalyzed by rubisco bring CO

to the level of a hexose,

converting CO

into a chemical fuel at the expense of NADPH and ATP generated from the light reactions.

The third phase of the Calvin cycle is the regeneration of ribulose 1,5-bisphosphate, the acceptor of CO

in the first step.

The problem is to construct a five-carbon sugar from six-carbon and three-carbon sugars. A transketolase and an aldolase

play the major role in the rearrangement of the carbon atoms. The transketolase, which we will see again in the pentose

phosphate pathway (Section 20.2.3), requires the coenzyme thiamine pyrophosphate (TPP) to transfer a two-carbon unit

(CO-CH

OH) from a ketose to an aldose.

We will consider the mechanism of transketolase when we meet it again in the pentose phosphate pathway (Section

20.3.2). Aldolase, which we have already encountered in glycolysis (Section 16.1.3), catalyzes an aldol condensation

between dihydroxyacetone phosphate and an aldehyde. This enzyme is highly specific for dihydroxyacetone phosphate,

but it accepts a wide variety of aldehydes.

With these enzymes, the construction of the five-carbon sugar proceeds as shown in Figure 20.10.