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

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(e) Galactose

(f) Dihydroxyacetone phosphate

See answer

Reference states. The standard oxidation-reduction potential for the reduction of O

to H

O is given as 0.82 V in

Table 18.1. However, the value given in textbooks of chemistry is 1.23 V. Account for this difference.

See answer

Potent poisons. What is the effect of each of the following inhibitors on electron transport and ATP formation by the

respiratory chain?

(a) Azide

(b) Atractyloside

(d) DNP

(e) Carbon monoxide

(f) Antimycin A

See answer

A question of coupling. What is the mechanistic basis for the observation that the inhibitors of ATP synthase also

lead to an inhibition of the electron-transport chain?

See answer

consumption. Using the axes in the adjoining illustration, draw an oxygen-uptake curve ([O

] versus time) for a

suspension of isolated mitochondria when the following compounds are added in the indicated order. With the

addition of each compound, all of the previously added compounds remain present. The experiment starts with the

amount of oxygen indicated by the arrow on the y-axis. [O

] can only decrease or be unaffected.

(a) Glucose

(b) ADP + P

(d) Oligomycin

(e) Succinate

(f) Dinitrophenol

(g) Rotenone

(h) Cyanide

See answer

P:O ratios. The number of molecules of inorganic phosphate incorporated into organic form per atom of oxygen

consumed, termed the P:O ratio, was frequently used as an index of oxidative phosphorylation.

(a) What is the relation of the P:O ratio to the ratio of the number of protons translocated per electron pair (H

/2 e

)

and the ratio of the number of protons needed to synthesize ATP and transport it to the cytosol (P/H

(b) What are the P:O ratios for electrons donated by matrix NADH and by succinate?

See answer

Thermodynamic constraint. Compare the ∆G°´ values for the oxidation of succinate by NAD

and by FAD. Use the

data given in Table 18.1, and assume that E´

for the FAD-FADH

redox couple is nearly 0 V. Why is FAD rather

than NAD

the electron acceptor in the reaction catalyzed by succinate dehydrogenase?

See answer

Cyanide antidote. The immediate administration of nitrite is a highly effective treatment for cyanide poisoning.

What is the basis for the action of this antidote? (Hint: Nitrite oxidizes ferrohemoglobin to ferrihemoglobin.)

See answer

Currency exchange. For a proton-motive force of 0.2 V (matrix negative), what is the maximum [ATP]/[ADP][P

]

ratio compatible with ATP synthesis? Calculate this ratio three times, assuming that the number of protons

translocated per ATP formed is two, three, and four and that the temperature is 25°C.

See answer

10.

Runaway mitochondria 1. Suppose that the mitochondria of a patient oxidizes NADH irrespective of whether ADP

is present. The P:O ratio for oxidative phosphorylation by these mitochondria is less than normal. Predict the likely

symptoms of this disorder.

See answer

11.

An essential residue. Conduction of protons by the F

unit of ATP synthase is blocked by modification of a single

side chain by dicyclohexylcarbodiimide. What are the most likely targets of action of this reagent? How might you

use site-specific mutagenesis to determine whether this residue is essential for proton conduction?

See answer

12.

Recycling device. The cytochrome b component of Q-cytochrome c oxidoreductase enables both electrons of QH

to be effectively utilized in generating a proton-motive force. Cite another recycling device in metabolism that

brings a potentially dead end reaction product back into the mainstream.

See answer

13.

Crossover point. The precise site of action of a respiratory-chain inhibitor can be revealed by the crossover

technique. Britton Chance devised elegant spectroscopic methods for determining the proportions of the oxidized

and reduced forms of each carrier. This determiniation is feasible because the forms have distinctive absorption

spectra, as illustrated in the adjoining graph for cytochrome c. You are given a new inhibitor and find that its

addition to respiring mitochondria causes the carriers between NADH and QH

to become more reduced and those

between cytochrome c and O

to become more oxidized. Where does your inhibitor act?

See answer

14.

Runaway mitochondria 2. Years ago, it was suggested that uncouplers would make wonderful diet drugs. Explain

why this idea was proposed and why it was rejected. Why might the producers of antiperspirants be supportive of

the idea?

See answer

15.

Identifying the inhibition. You are asked to determine whether a chemical is an electron-transport-chain inhibitor or

an inhibitor of ATP synthase. Design an experiment to determine this.

See answer

Chapter Integration Problem

16.

No exchange. Mice that are completely lacking adenine nucleotide translocase (ANT

/ANT

) can be made by the

"knockout" technique. Remarkably, these mice are viable but have the following pathological conditions: (1) high

serum levels of lactate, alanine, and succinate; (2) there is little electron transport; and (3) the levels of

mitochondrial H

are increased six- to eightfold compared with normal mice. Provide a possible biochemical

explanation for each of these conditions.

See answer

Data Interpretation Problem

17.

Mitochondrial disease. A mutation in a mitochondrial gene encoding a component of ATP synthase has been

identified. People who have this mutation suffer from muscle weakness, ataxia, and retinitis pigmentosa. A tissue

biopsy was performed on each patient, and submitochondrial particles were isolated that were capable of succinate-

sustained ATP synthesis. First, the activity of the ATP synthase was measured on the addition of succinate and the

following results were obtained.

(a) What was the purpose of the addition of succinate?

(b) What is the effect of the mutation on succinate-coupled ATP synthesis?

Next, the ATPase activity of the enzyme was measured by incubating the submitochondrial particle with ATP in

the absence of succinate.

(d) What is the effect of the mutation on ATP hydrolysis?

(e) What do these results, in conjunction with those obtained in the first experiment, tell you about the nature of the

mutation?

See answer

Mechanism Problem

18.

Chiral clue. ATPγS, a slowly hydrolyzed analog of ATP, can be used to probe the mechanism of phosphoryl

transfer reactions. Chiral ATPγS has been synthesized containing

O in a specific γ position and ordinary

elsewhere in the molecule. Hydrolysis of this chiral molecule by ATP synthase in

O-enriched water yields

inorganic [

O]thiophosphate having the following absolute configuration. In contrast, hydrolysis of this

chiral ATPγS by a calcium-pumping ATPase from muscle gives thiophosphate of the opposite configuration. What

is the simplest interpretation of these data?

See answer

Media Problem

19.

Queueing up? The Conceptual Insights module on oxidative phophorylation summarizes the steps in the

electron transport chain and can help you answer the following questions. Suppose you have isolated actively

respiring mitochondria. What do you think might happen to ATP production in the short-term if you were to add

large amounts of coenzyme Q? If you were to add large amounts of QH

? How might the long-term responses

differ from the short-term?

II. Transducing and Storing Energy 18. Oxidative Phosphorylation

Selected Readings

Where to start

M.W. Gray, G. Burger, and B.F. Lang. 1999. Mitochondrial evolution Science 283: 1476-1481. (PubMed)

D.C. Wallace. 1997. Mitochondrial DNA in aging and disease Sci. Am. 277: (2) 40-47. (PubMed)

M. Saraste. 1999. Oxidative phosphorylation at the fin de siècle Science 283: 1488-1493. (PubMed)

B.E. Shultz and S.I. Chan. 2001. Structures and proton-pumping strategies of mitochondrial respiratory enzymes Ann.

Rev. Biophys. Biomol. Struct. 30: 23-65.

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)

Books

Scheffler, I. E., 1999. Mitochondria. Wiley.

Nicholls, D. G., and Ferguson, S. J., 1997. Bioenergetics 2. Academic Press.

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

Ernster, L. (Ed.), 1992. Molecular Mechanisms in Bioenergetics. Elsevier.

Electron-transport chain

D. Zaslavsky and R.B. Gennis. 2000. Proton pumping by cytochrome oxidase: Progress, problems and postulates

Biochim. Biophys. Acta 1458: 164-179. (PubMed)

N. Grigorieff. 1999. Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I) Curr. Opin. Struct. Biol.

9: 476-483. (PubMed)

B.A. Ackrell. 2000. Progress in understanding structure-function relationships in respiratory chain complex II FEBS Lett.

466: 1-5. (PubMed)

N. Grigorieff. 1998. Three-dimensional structure of bovine NADH: ubiquinone oxidoreductase (complex I) at 22 Å in

ice J. Mol. Biol. 277: 1033-1046. (PubMed)

P.L. Dutton, C.C. Moser, V.D. Sled, F. Daldal, and T. Ohnishi. 1998. A reductant-induced oxidation mechanism for

complex I Biochim. Biophys. Acta 1364: 245-257. (PubMed)

Z. Zhang, L. Huang, V.M. Shulmeister, Y.I. Chi, K.K. Kim, L.W. Hung, A.R. Crofts, E.A. Berry, and S.H. Kim. 1998.

Electron transfer by domain movement in cytochrome bc

Nature 392: 677-684. (PubMed)

D. Xia, C.A. Yu, H. Kim, J.Z. Xia, A.M. Kachurin, L. Zhang, L. Yu, and J. Deisenhofer. 1997. Crystal structure of the

cytochrome bc

complex from bovine heart mitochondria Science 277: 60-66. (PubMed)

H. Michel, J. Behr, A. Harrenga, and A. Kannt. 1998. Cytochrome c oxidase: Structure and spectroscopy Annu. Rev.

Biophys. Biomol. Struct. 27: 329-356. (PubMed)

M.I. Verkhovsky, A. Jasaitis, M.L. Verkhovskaya, J.E. Morgan, and M. Wikstrom. 1999. Proton translocation by

cytochrome c oxidase Nature 400: 480-483. (PubMed)

M. Wikstrom and J.E. Morgan. 1992. The dioxygen cycle: Spectral, kinetic, and thermodynamic characteristics of ferryl

and peroxy intermediates observed by reversal of the cytochrome oxidase reaction J. Biol. Chem. 267: 10266-10273.

(PubMed)

ATP synthase

H. Noji and M. Yoshida. 2001. The rotary machine in the cell: ATP synthase J. Biol. Chem. 276: 1665-1668. (PubMed)

R. Yasuda, H. Noji, K. Kinosita Jr, and M. Yoshida. 1998. F1- ATPase is a highly efficient molecular motor that rotates

with discrete 120 degree steps Cell 93: 1117-1124. (PubMed)

K. Kinosita Jr, R. Yasuda, H. Noji, S. shiwata, and M. Yoshida. 1998. F1-ATPase: A rotary motor made of a single

molecule Cell 93: 21-24. (PubMed)

H. Noji, R. Yasuda, M. Yoshida, and K. Kinosita Jr. 1997. Direct observation of the rotation of F1-ATPase Nature 386:

299-302. (PubMed)

S.P. Tsunoda, R. Aggeler, M. Yoshida, and R.A. Capaldi. 2001. Rotation of the c subunit oligomer in fully functional F

ATP synthase Proc. Natl. Acad. Sci. USA 987: 898-902. (PubMed)

C. Gibbons, M.G. Montgomery, A.G.W. Leslie, and J. Walker. 2000. The structure of the central stock in F

-ATPase at

2.4 Å resolution Nat. Struct. Biol. 7: 1055-1061. (PubMed)

P.D. Boyer. 2000. Catalytic site forms and controls in ATP synthase catalysis Biochim. Biophys. Acta 1458: 252-262.

(PubMed)

D. Stock, A.G. Leslie, and J.E. Walker. 1999. Molecular architecture of the rotary motor in ATP synthase Science 286:

1700-1705. (PubMed)

Y. Sambongi, Y. Iko, M. Tanabe, H. Omote, A. Iwamoto-Kihara, I. Ueda, T. Yanagida, Y. Wada, and M. Futai. 1999.

Mechanical rotation of the c subunit oligomer in ATP synthase (F

): Direct observation Science 286: 1722-1724.

(PubMed)

J.P. Abrahams, A.G. Leslie, R. Lutter, and J.E. Walker. 1994. Structure at 2.8 Å resolution of F1-ATPase from bovine

heart mitochondria Nature 370: 621-628. (PubMed)

M.A. Bianchet, J. Hullihen, P.L. Pedersen, and L.M. Amzel. 1998. The 2.8-Å structure of rat liver F1-ATPase:

Configuration of a critical intermediate in ATP synthesis/hydrolysis Proc. Natl. Acad. Sci. USA 95: 11065-11070.

(PubMed) (Full Text in PMC)

Translocators

M. Klingenberg and S.G. Huang. 1999. Structure and function of the uncoupling protein from brown adipose tissue

Biochim. Biophys. Acta 1415: 271-296. (PubMed)

D.G. Nicholls and E. Rial. 1999. A history of the first uncoupling protein, UCP1 J. Bioenerg. Biomembr. 31: 399-406.

(PubMed)

D. Ricquier and F. Bouillaud. 2000. The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP

Biochem. J. 345: 161-179. (PubMed)

J.E. Walker. 1992. The mitochondrial transporter family Curr. Opin. Struct. Biol. 2: 519-526.

M. Klingenberg. 1992. Structure-function of the ADP/ATP carrier Biochem. Soc. Trans. 20: 547-550. (PubMed)

Superoxide dismutase and catalase

V.C. Culotta. 2000. Superoxide dismutase, oxidative stress, and cell metabolism Curr. Top. Cell Regul. 36: 117-132.

(PubMed)

B.M. Morrison, J.H. Morrison, and J.W. Gordon. 1998. Superoxide dismutase and neurofilament transgenic models of

amyotrophic lateral sclerosis J. Exp. Zool. 282: 32-47. (PubMed)

J.A. Tainer, E.D. Getzoff, J.S. Richardson, and D.C. Richardson. 1983. Structure and mechanism of copper, zinc

superoxide dismutase Nature 306: 284-287. (PubMed)

T.J. Reid, M.R. Murthy, A. Sicignano, N. Tanaka, W.D. Musick, and M.G. Rossmann. 1981. Structure and heme

environment of beef liver catalase at 2.5 Å resolution Proc. Natl. Acad. Sci. USA 78: 4767-4771. (PubMed)

W.C. Stallings, K.A. Pattridge, R.K. Strong, and M.L. Ludwig. 1984. Manganese and iron superoxide dismutases are

structural homologs J. Biol. Chem. 259: 10695-10699. (PubMed)

Y. Hsieh, Y. Guan, C. Tu, P.J. Bratt, A. Angerhofer, J.R. Lepock, M.J. Hickey, J.A. Tainer, H.S. Nick, and D.N.

Silverman. 1998. Probing the active site of human manganese superoxide dismutase: The role of glutamine 143

Biochemistry 37: 4731-4739. (PubMed)

Mitochondrial diseases

J. Smeitink, L. van den Heuvel, and S. DiMauro. 2001. The genetics and pathology of oxidative phosphorylation Nature

Rev. Genet 2: 342-352.

D.C. Wallace. 1999. Mitochondrial diseases in man and mouse Science 283: 1482-1488. (PubMed)

D.C. Wallace. 1992. Diseases of the mitochondrial DNA Annu. Rev. Biochem. 61: 1175-1212. (PubMed)

R. Benecke, P. Strumper, and H. Weiss. 1992. Electron transfer Complex I defect in idiopathic dystonia Ann. Neurol. 32:

683-686. (PubMed)

Apoptosis

N. Joza, S.A. Susin, E. Daugas, W.L. Stanford, S.K. Cho, C.Y.J. Li, T. Sasaki, A.J. Elia, H.-Y.M. Cheng, L. Ravagnan,

K.F. Ferri, N. Zamzami, A. Wakeham, R. Hakem, H. Yoshida, Y.-Y. Kong, T.W. Mak, J.C. Zúñiga-Pflücker, G.

Kroemer, and J.M. Penninger. 2001. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell

death Nature 410: 549-554. (PubMed)

S. Desagher and J.C. Martinou. 2000. Mitochondria as the central control point of apoptosis Trends Cell Biol. 10: 369-

377. (PubMed)

M.O. Hengartner. 2000. The biochemistry of apoptosis Nature 407: 770-776. (PubMed)

W.C. Earnshaw, L.M. Martins, and S.H. Kaufmann. 1999. Mammalian caspases: Structure, activation, substrates, and

functions during apoptosis Annu. Rev. Biochem. 68: 383-424. (PubMed)

B.B. Wolf and D.R. Green. 1999. Suicidal tendencies: Apoptotic cell death by caspase family proteinases J. Biol. Chem.

274: 20049-20052. (PubMed)

Historical aspects

P. Mitchell. 1979. Keilin's respiratory chain concept and its chemiosmotic consequences Science 206: 1148-1159.

(PubMed)

P. Mitchell. 1976. Vectorial chemistry and the molecular mechanics of chemiosmotic coupling: Power transmission by

proticity Biochem. Soc. Trans. 4: 399-430. (PubMed)

E. Racker. 1980. From Pasteur to Mitchell: A hundred years of bioenergetics Fed. Proc. 39: 210-215. (PubMed)

Keilin, D., 1966. The History of Cell Respiration and Cytochromes. Cambridge University Press.

Kalckar, H. M. (Ed.), 1969. Biological Phosphorylations: Development of Concepts. Prentice Hall.

H.M. Kalckar. 1991. Fifty years of biological research: From oxidative phosphorylation to energy requiring transport and

regulation Annu. Rev. Biochem. 60: 1-37. (PubMed)

Fruton, J. S., 1972. Molecules and Life: Historical Essays on the Interplay of Chemistry and Biology. Wiley-

Interscience.

II. Transducing and Storing Energy

19. The Light Reactions of Photosynthesis

Essentially all free energy utilized by biological systems arises from solar energy that is trapped by the process of

photosynthesis. The basic equation of photosynthesis is deceptively simple. Water and carbon dioxide combine to form

carbohydrates and molecular oxygen.

In this equation, (CH

O) represents carbohydrate, primarily sucrose and starch. The mechanism of photosynthesis is

complex and requires the interplay of many proteins and small molecules. Photosynthesis in green plants takes place in

chloroplasts (Figure 19.1). The energy of light captured by pigment molecules, called chlorophylls, in chloroplasts is

used to generate high-energy electrons with great reducing potential. These electrons are used to produce NADPH as

well as ATP in a series of reactions called the light reactions because they require light. NADPH and ATP formed by the

action of light then reduce carbon dioxide and convert it into 3-phosphoglycerate by a series of reactions called the

Calvin cycle or the dark reactions. The Calvin cycle will be discussed in Chapter 20. The amount of energy stored by

photosynthesis is enormous. More than 10

kcal (4.2 × 10

kJ) of free energy is stored annually by photosynthesis on

Earth, which corresponds to the assimilation of more than 10

tons of carbon into carbohydrate and other forms of

organic matter.

As animals ourselves, we perhaps easily overlook the ultimate primacy of photosynthesis for our biosphere.

Photosynthesis is the source of essentially all the carbon compounds and all the oxygen that makes aerobic metabolism

possible. Moreover, as we shall see, there are considerable mechanistic and evolutionary parallels between the light

reactions of photosynthesis and steps in oxidative phosphorylation.

19.0.1. Photosynthesis: An Overview

We can use our understanding of the citric acid cycle and oxidative phosphorylation to anticipate the processes required

of photosynthesis. The citric acid cycle oxidizes carbon fuels to CO

to generate high-energy electrons, notably in the

form of NADH. The flow of these high-energy electrons generates a proton-motive force through the action of the

electron-transport chain. This proton-motive force is then transduced by ATP synthase to form ATP. A principal

difference between oxidative phosphorylation and photosynthesis is the source of the high-energy electrons. The light

reactions of photosynthesis use energy from photons to generate high-energy electrons (Figure 19.2). These electrons are

used directly to reduce NADP

to NADPH and are used indirectly through an electron-transport chain to generate a

proton-motive force across a membrane. A side product of these reactions is O

. The proton-motive force drives ATP

synthesis through the action of an ATP synthase, homologous to that in oxidative phosphorylation. In the dark reactions,

the NADPH and ATP formed by the action of light drive the reduction of CO

to more-useful organic compounds.

Photosynthetic yield-

"If a year's yield of photosynthesis were amassed in the form of

sugar cane, it would form a heap over two miles high and with a base

43 square miles."

-G. E. F

ogge

If all of this sugar cane were converted into sugar cubes (0.5 inch on

a side) and stacked end to end, the sugar cubes would extend 1.6 ×

miles, or to the planet Pluto.

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

Figure 19.1. Electron Micrograph of a Chloroplast from a Spinach Leaf. The thylakoid membranes pack together to

form grana. [Courtesy of Dr. Kenneth Miller.]

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

Figure 19.2. The Light Reactions of Photosynthesis. Light is absorbed and the energy is used to drive electrons from

water to generate NADPH and to drive protons across a membrane. These protons return through ATP synthase to make

ATP.

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