Oxidative
Phosphorylation and
Photophosphorylation
chapter
19
1. Oxidation-Reduction Reactions The NADH dehydrogenase complex of the mitochondrial respira-
tory chain promotes the following series of oxidation-reduction reactions, in which Fe
3
and Fe
2
represent the iron in iron-sulfur centers, Q is ubiquinone, QH
2
is ubiquinol, and E is the enzyme:
(1) NADH H
E-FMN 88n NAD
E-FMNH
2
(2) E-FMNH
2
2Fe
3
88n E-FMN 2Fe
2
2H
(3) 2Fe
2
2H
Q88n 2Fe
3
QH
2
Sum: NADH H
Q88n NAD
QH
2
For each of the three reactions catalyzed by the NADH dehydrogenase complex, identify (a) the
electron donor, (b) the electron acceptor, (c) the conjugate redox pair, (d) the reducing agent, and
(e) the oxidizing agent.
2. All Parts of Ubiquinone Have a Function In electron transfer, only the quinone portion of
ubiquinone undergoes oxidation-reduction; the isoprenoid side chain remains unchanged. What is
the function of this chain?
c19OxidativePhosphorylationandPhotophosphorylation.qxd 11/30/12 7:15 PM Page S-223
S-224 Chapter 19 Oxidative Phosphorylation and Photophosphorylation
3. Use of FAD Rather Than NAD
ⴙ
in Succinate Oxidation All the dehydrogenases of glycolysis and
the citric acid cycle use NAD
(E for NAD
/NADH is 0.32 V) as electron acceptor except succinate
dehydrogenase, which uses covalently bound FAD (E for FAD/FADH
2
in this enzyme is 0.050 V).
Suggest why FAD is a more appropriate electron acceptor than NAD
in the dehydrogenation of succi-
nate, based on the E values of fumarate/succinate (E 0.031), NAD
/NADH, and the succinate
dehydrogenase FAD/FADH
2
.
4. Degree of Reduction of Electron Carriers in the Respiratory Chain The degree of reduction of
each carrier in the respiratory chain is determined by conditions in the mitochondrion. For example,
when NADH and O
2
are abundant, the steady-state degree of reduction of the carriers decreases as
electrons pass from the substrate to O
2
. When electron transfer is blocked, the carriers before the block
become more reduced and those beyond the block become more oxidized (see Fig. 19–6). For each of the
conditions below, predict the state of oxidation of ubiquinone and cytochromes b, c
1
, c, and aa
3
.
(a) Abundant NADH and O
2
, but cyanide added
(b) Abundant NADH, but O
2
exhausted
(c) Abundant O
2
, but NADH exhausted
(d) Abundant NADH and O
2
5. Effect of Rotenone and Antimycin A on Electron Transfer Rotenone, a toxic natural product
from plants, strongly inhibits NADH dehydrogenase of insect and fish mitochondria. Antimycin A, a
toxic antibiotic, strongly inhibits the oxidation of ubiquinol.
(a) Explain why rotenone ingestion is lethal to some insect and fish species.
(b) Explain why antimycin A is a poison.
(c) Given that rotenone and antimycin A are equally effective in blocking their respective sites in the
electron-transfer chain, which would be a more potent poison? Explain.
Answer
6. Uncouplers of Oxidative Phosphorylation In normal mitochondria the rate of electron transfer is
tightly coupled to the demand for ATP. When the rate of use of ATP is relatively low, the rate of
electron transfer is low; when demand for ATP increases, electron-transfer rate increases. Under these
conditions of tight coupling, the number of ATP molecules produced per atom of oxygen consumed
when NADH is the electron donor—the P/O ratio–is about 2.5.
(a) Predict the effect of a relatively low and a relatively high concentration of uncoupling agent on
the rate of electron transfer and the P/O ratio.
(b) Ingestion of uncouplers causes profuse sweating and an increase in body temperature. Explain
this phenomenon in molecular terms. What happens to the P/O ratio in the presence of uncou-
plers?
(c) The uncoupler 2,4-dinitrophenol was once prescribed as a weight-reducing drug. How could this
agent, in principle, serve as a weight-reducing aid? Uncoupling agents are no longer prescribed
because some deaths occurred following their use. Why might the ingestion of uncouplers lead to
death?
7. Effects of Valinomycin on Oxidative Phosphorylation When the antibiotic valinomycin is added
to actively respiring mitochondria, several things happen: the yield of ATP decreases, the rate of O
2
consumption increases, heat is released, and the pH gradient across the inner mitochondrial mem-
brane increases. Does valinomycin act as an uncoupler or an inhibitor of oxidative phosphorylation?
Explain the experimental observations in terms of the antibiotic’s ability to transfer K
ions across the
inner mitochondrial membrane.
Chapter 19 Oxidative Phosphorylation and Photophosphorylation S-225
c19OxidativePhosphorylationandPhotophosphorylation.qxd 11/30/12 7:15 PM Page S-225
S-226 Chapter 19 Oxidative Phosphorylation and Photophosphorylation
8. Mode of Action of Dicyclohexylcarbodiimide (DCCD) When DCCD is added to a suspension of
tightly coupled, actively respiring mitochondria, the rate of electron transfer (measured by O
2
con-
sumption) and the rate of ATP production dramatically decrease. If a solution of 2,4-dinitrophenol is
now added to the preparation, O
2
consumption returns to normal but ATP production remains inhibited.
(a) What process in electron transfer or oxidative phosphorylation is affected by DCCD?
(b) Why does DCCD affect the O
2
consumption of mitochondria? Explain the effect of 2,4-dinitro-
phenol on the inhibited mitochondrial preparation.
(c) Which of the following inhibitors does DCCD most resemble in its action: antimycin A, rotenone,
or oligomycin?
Answer
9. Compartmentalization of Citric Acid Cycle Components Isocitrate dehydrogenase is found only
in the mitochondrion, but malate dehydrogenase is found in both the cytosol and mitochondrion. What
is the role of cytosolic malate dehydrogenase?
10. The Malate–a-Ketoglutarate Transport System The transport system that conveys malate
and -ketoglutarate across the inner mitochondrial membrane (see Fig. 19–31) is inhibited by n–
butylmalonate. Suppose n-butylmalonate is added to an aerobic suspension of kidney cells using glucose
exclusively as fuel. Predict the effect of this inhibitor on (a) glycolysis, (b) oxygen consumption,
(c) lactate formation, and (d) ATP synthesis.
11. Cellular ADP Concentration Controls ATP Formation Although ADP and P
i
are required for the
synthesis of ATP, the rate of synthesis depends mainly on the concentration of ADP, not P
i
. Why?
12. Time Scales of Regulatory Events in Mitochondria Compare the likely time scales for the adjust-
ments in respiratory rate caused by (a) increased [ADP] and (b) reduced pO
2
. What accounts for the
difference?
13. The Pasteur Effect When O
2
is added to an anaerobic suspension of cells consuming glucose at a
high rate, the rate of glucose consumption declines greatly as the O
2
is used up, and accumulation of
lactate ceases. This effect, first observed by Louis Pasteur in the 1860s, is characteristic of most cells
capable of aerobic and anaerobic glucose catabolism.
(a) Why does the accumulation of lactate cease after O
2
is added?
(b) Why does the presence of O
2
decrease the rate of glucose consumption?
(c) How does the onset of O
2
consumption slow down the rate of glucose consumption? Explain in
terms of specific enzymes.
Chapter 19 Oxidative Phosphorylation and Photophosphorylation S-227
c19OxidativePhosphorylationandPhotophosphorylation.qxd 11/30/12 7:15 PM Page S-227
S-228 Chapter 19 Oxidative Phosphorylation and Photophosphorylation
14. Respiration-Deficient Yeast Mutants and Ethanol Production Respiration-deficient yeast mu-
tants (p
; “petites”) can be produced from wild-type parents by treatment with mutagenic agents. The
mutants lack cytochrome oxidase, a deficit that markedly affects their metabolic behavior. One striking
effect is that fermentation is not suppressed by O
2
—that is, the mutants lack the Pasteur effect (see
Problem 13). Some companies are very interested in using these mutants to ferment wood chips to
ethanol for energy use. Explain the advantages of using these mutants rather than wild-type yeast for
large-scale ethanol production. Why does the absence of cytochrome oxidase eliminate the Pasteur effect?
15. Advantages of Supercomplexes for Electron Transfer There is growing evidence that mitochon-
drial Complexes I, II, III, and IV are part of a larger supercomplex. What might be the advantage of
having all four complexes within a supercomplex?
16. How Many Protons in a Mitochondrion? Electron transfer translocates protons from the mito-
chondrial matrix to the external medium, establishing a pH gradient across the inner membrane
(outside more acidic than inside). The tendency of protons to diffuse back into the matrix is the driving
force for ATP synthesis by ATP synthase. During oxidative phosphorylation by a suspension of mito-
chondria in a medium of pH 7.4, the pH of the matrix has been measured as 7.7.
(a) Calculate [H
] in the external medium and in the matrix under these conditions.
(b) What is the outside-to-inside ratio of [H
]? Comment on the energy inherent in this concentration
difference. (Hint: see Eqn 11–4)
(c) Calculate the number of protons in a respiring liver mitochondrion, assuming its inner matrix
compartment is a sphere of diameter 1.5 mm.
(d) From these data, is the pH gradient alone sufficient to generate ATP?
(e) If not, suggest how the necessary energy for synthesis of ATP arises.
Answer
17. Rate of ATP Turnover in Rat Heart Muscle Rat heart muscle operating aerobically fills more than
90% of its ATP needs by oxidative phosphorylation. Each gram of tissue consumes O
2
at the rate of
10.0 mmol/min, with glucose as the fuel source.
(a) Calculate the rate at which the heart muscle consumes glucose and produces ATP.
(b) For a steady-state concentration of ATP of 5.0 mmol/g of heart muscle tissue, calculate the time
required (in seconds) to completely turn over the cellular pool of ATP. What does this result indi-
cate about the need for tight regulation of ATP production? (Note: Concentrations are expressed
as micromoles per gram of muscle tissue because the tissue is mostly water.)
18. Rate of ATP Breakdown in Flight Muscle ATP production in the flight muscle of the fly Lucilia
sericata results almost exclusively from oxidative phosphorylation. During flight, 187 mL of O
2
/hr ⴢg
of body weight is needed to maintain an ATP concentration of 7.0 mol/g of flight muscle. Assuming
that flight muscle makes up 20% of the weight of the fly, calculate the rate at which the flight-muscle
ATP pool turns over. How long would the reservoir of ATP last in the absence of oxidative phosphory-
lation? Assume that reducing equivalents are transferred by the glycerol 3-phosphate shuttle and that
O
2
is at 25 C and 101.3 kPa (1 atm).
19. Mitochondrial Disease and Cancer Mutations in the genes that encode certain mitochondrial pro-
teins are associated with a high incidence of some types of cancer. How might defective mitochondria
lead to cancer?
Chapter 19 Oxidative Phosphorylation and Photophosphorylation S-229
c19OxidativePhosphorylationandPhotophosphorylation.qxd 12/7/12 7:25 PM Page S-229
S-230 Chapter 19 Oxidative Phosphorylation and Photophosphorylation
20. Variable Severity of a Mitochondrial Disease Individuals with a disease caused by a specific de-
fect in the mitochondrial genome may have symptoms ranging from mild to severe. Explain why.
21. Transmembrane Movement of Reducing Equivalents Under aerobic conditions, extramitochon-
drial NADH must be oxidized by the mitochondrial electron-transfer chain. Consider a preparation of
rat hepatocytes containing mitochondria and all the cytosolic enzymes. If [4-
3
H]NADH is introduced,
radioactivity soon appears in the mitochondrial matrix. However, if [7-
14
C]NADH is introduced, no
radioactivity appears in the matrix. What do these observations reveal about the oxidation of extramito-
chondrial NADH by the electron-transfer chain?
O
14
C
H
N
NH
2
R
[7-
14
C]NADH
O
3
HC
N
3
H
NH
2
R
[4-
3
H]NADH
H
22. High Blood Alanine Level Associated with Defects in Oxidative Phosphorylation Most
individuals with genetic defects in oxidative phosphorylation are found to have relatively high
concentrations of alanine in their blood. Explain this in biochemical terms.
23. NAD Pools and Dehydrogenase Activities Although both pyruvate dehydrogenase and glyceralde-
hyde 3-phosphate dehydrogenase use NAD
as their electron acceptor, the two enzymes do not com-
pete for the same cellular NAD pool. Why?
24. Diabetes as a Consequence of Mitochondrial Defects Glucokinase is essential in the metabolism
of glucose in pancreatic cells. Humans with two defective copies of the glucokinase gene exhibit a
severe, neonatal diabetes, whereas those with only one defective copy of the gene have a much milder
form of the disease (mature onset diabetes of the young, MODY2). Explain this difference in terms of
the biology of the cell.
25. Effects of Mutations in Mitochondrial Complex II Single nucleotide changes in the gene for suc-
cinate dehydrogenase (Complex II) are associated with midgut carcinoid tumors. Suggest a mecha-
nism to explain this observation.
26. Photochemical Efficiency of Light at Different Wavelengths The rate of photosynthesis, mea-
sured by O
2
production, is higher when a green plant is illuminated with light of wavelength 680 nm
than with light of 700 nm. However, illumination by a combination of light of 680 nm and 700 nm gives
a higher rate of photosynthesis than light of either wavelength alone. Explain.
27. Balance Sheet for Photosynthesis In 1804 Theodore de Saussure observed that the total weights of
oxygen and dry organic matter produced by plants is greater than the weight of carbon dioxide con-
sumed during photosynthesis. Where does the extra weight come from?
28. Role of H
2
S in Some Photosynthetic Bacteria Illuminated purple sulfur bacteria carry out photo-
synthesis in the presence of H
2
O and
14
CO
2
, but only if H
2
S is added and O
2
is absent. During the
course of photosynthesis, measured by formation of [
14
C]carbohydrate, H
2
S is converted to elemental
sulfur, but no O
2
is evolved. What is the role of the conversion of H
2
S to sulfur? Why is no O
2
evolved?
Chapter 19 Oxidative Phosphorylation and Photophosphorylation S-231
c19OxidativePhosphorylationandPhotophosphorylation.qxd 12/7/12 7:25 PM Page S-231
S-232 Chapter 19 Oxidative Phosphorylation and Photophosphorylation
29. Boosting the Reducing Power of Photosystem I by Light Absorption When photosystem I
absorbs red light at 700 nm, the standard reduction potential of P700 changes from 0.40 V to about
1.2 V. What fraction of the absorbed light is trapped in the form of reducing power?
30. Electron Flow through Photosystems I and II Predict how an inhibitor of electron passage
through pheophytin would affect electron flow through (a) photosystem II and (b) photosystem I.
Explain your reasoning.
31. Limited ATP Synthesis in the Dark In a laboratory experiment, spinach chloroplasts are illumi-
nated in the absence of ADP and P
i
, then the light is turned off and ADP and P
i
are added. ATP is syn-
thesized for a short time in the dark. Explain this finding.
32. Mode of Action of the Herbicide DCMU When chloroplasts are treated with 3-(3,4-dichlorophenyl)-
1,1-dimethylurea (DCMU, or diuron), a potent herbicide, O
2
evolution and photophosphorylation
cease. Oxygen evolution, but not photophosphorylation, can be restored by addition of an external
electron acceptor, or Hill reagent. How does DCMU act as a weed killer? Suggest a location for the in-
hibitory action of this herbicide in the scheme shown in Figure 19–58. Explain.
33. Effect of Venturicidin on Oxygen Evolution Venturicidin is a powerful inhibitor of the chloroplast
ATP synthase, interacting with the CF
o
part of the enzyme and blocking proton passage through the
CF
o
CF
1
complex. How would venturicidin affect oxygen evolution in a suspension of well-illuminated
chloroplasts? Would your answer change if the experiment were done in the presence of an uncou-
pling reagent such as 2,4-dinitrophenol (DNP)? Explain.
34. Bioenergetics of Photophosphorylation The steady-state concentrations of ATP, ADP, and P
i
in
isolated spinach chloroplasts under full illumination at pH 7.0 are 120.0, 6.0, and 700.0 m
M
, respectively.
(a) What is the free-energy requirement for the synthesis of 1 mol of ATP under these conditions?
(b) The energy for ATP synthesis is furnished by light-induced electron transfer in the chloroplasts.
What is the minimum voltage drop necessary (during transfer of a pair of electrons) to synthe-
size ATP under these conditions? (You may need to refer to Eqn 13–7.)
Answer
35. Light Energy for a Redox Reaction Suppose you have isolated a new photosynthetic microorgan-
ism that oxidizes H
2
S and passes the electrons to NAD
. What wavelength of light would provide
enough energy for H
2
S to reduce NAD
under standard conditions? Assume 100% efficiency in the
photochemical event, and use E of 243 mV for H
2
S and 320 mV for NAD
. See Figure 19–48 for
the energy equivalents of wavelengths of light.
S-234 Chapter 19 Oxidative Phosphorylation and Photophosphorylation
36. Equilibrium Constant for Water-Splitting Reactions The coenzyme NADP
is the terminal elec-
tron acceptor in chloroplasts, according to the reaction
2H
2
O 2NADP
88n 2NADPH 2H
O
2
Use the information in Table 19–2 to calculate the equilibrium constant for this reaction at 25 C. (The
relationship between K
eq
and G is discussed on p. 508.) How can the chloroplast overcome this un-
favorable equilibrium?
37. Energetics of Phototransduction During photosynthesis, eight photons must be absorbed (four by
each photosystem) for every O
2
molecule produced:
2H
2
O 2NADP
8 photons 88n 2NADPH 2H
O
2
Assuming that these photons have a wavelength of 700 nm (red) and that the absorption and use of
light energy are 100% efficient, calculate the free-energy change for the process.
38. Electron Transfer to a Hill Reagent Isolated spinach chloroplasts evolve O
2
when illuminated in
the presence of potassium ferricyanide (a Hill reagent), according to the equation
2H
2
O 4Fe
3
88n O
2
4H
4Fe
2
where Fe
3
represents ferricyanide and Fe
2
, ferrocyanide. Is NADPH produced in this process?
Explain.
39. How Often Does a Chlorophyll Molecule Absorb a Photon? The amount of chlorophyll a(M
r
892)
in a spinach leaf is about 20 mg/cm
2
of leaf. In noonday sunlight (average energy reaching the leaf is
5.4 J/cm
2
ⴢmin), the leaf absorbs about 50% of the radiation. How often does a single chlorophyll mol-
ecule absorb a photon? Given that the average lifetime of an excited chlorophyll molecule in vivo is
1 ns, what fraction of the chlorophyll molecules are excited at any one time?
40. Effect of Monochromatic Light on Electron Flow The extent to which an electron carrier is oxi-
dized or reduced during photosynthetic electron transfer can sometimes be observed directly with a
spectrophotometer. When chloroplasts are illuminated with 700 nm light, cytochrome f, plastocyanin,
and plastoquinone are oxidized. When chloroplasts are illuminated with 680 nm light, however, these
electron carriers are reduced. Explain.
41. Function of Cyclic Photophosphorylation When the [NADPH]/[NADP
] ratio in chloroplasts is high,
photophosphorylation is predominantly cyclic (see Fig. 19–58). Is O
2
evolved during cyclic photophos-
phorylation? Is NADPH produced? Explain. What is the main function of cyclic photophosphorylation?
Data Analysis Problem
42. Photophosphorylation: Discovery, Rejection, and Rediscovery In the 1930s and 1940s, re-
searchers were beginning to make progress toward understanding the mechanism of photosynthesis.
At the time, the role of “energy-rich phosphate bonds” (today, “ATP”) in glycolysis and cellular respi-
ration was just becoming known. There were many theories about the mechanism of photosynthesis,
especially about the role of light. This problem focuses on what was then called the “primary photo-
chemical process”—that is, on what it is, exactly, that the energy from captured light produces in the
photosynthetic cell. Interestingly, one important part of the modern model of photosynthesis was pro-
posed early on, only to be rejected, ignored for several years, then finally revived and accepted.
Chapter 19 Oxidative Phosphorylation and Photophosphorylation S-235
c19OxidativePhosphorylationandPhotophosphorylation.qxd 12/7/12 7:25 PM Page S-235
S-236 Chapter 19 Oxidative Phosphorylation and Photophosphorylation
In 1944, Emerson, Stauffer, and Umbreit proposed that “the function of light energy in photosyn-
thesis is the formation of ‘energy-rich’ phosphate bonds” (p. 107). In their model (hereafter, the
“Emerson model”), the free energy necessary to drive both CO
2
fixation and reduction came from
these “energy-rich phosphate bonds” (i.e., ATP), produced as a result of light absorption by a chloro-
phyll-containing protein.
This model was explicitly rejected by Rabinowitch (1945). After summarizing Emerson and coau-
thors’ findings, Rabinowitch stated: “Until more positive evidence is provided, we are inclined to con-
sider as more convincing a general argument against this hypothesis, which can be derived from en-
ergy considerations. Photosynthesis is eminently a problem of energy accumulation. What good can
be served, then, by converting light quanta (even those of red light, which amount to about 43 kcal per
Einstein) into ‘phosphate quanta’ of only 10 kcal per mole? This appears to be a start in the wrong
direction—toward dissipation rather than toward accumulation of energy” (Vol. I, p. 228). This argu-
ment, along with other evidence, led to the abandonment of the Emerson model until the 1950s, when
it was found to be correct—albeit in a modified form.
For each piece of information from Emerson and coauthors’ article presented in (a) through (d)
below, answer the following three questions:
1. How does this information support the Emerson model, in which light energy is used directly by
chlorophyll to make ATP, and the ATP then provides the energy to drive CO
2
fixation and
reduction?
2. How would Rabinowitch explain this information, based on his model (and most other models of
the day), in which light energy is used directly by chlorophyll to make reducing compounds?
Rabinowitch wrote: “Theoretically, there is no reason why all electronic energy contained in
molecules excited by the absorption of light should not be available for oxidation-reduction” (Vol. I,
p. 152). In this model, the reducing compounds are then used to fix and reduce CO
2
, and the en-
ergy for these reactions comes from the large amounts of free energy released by the reduction
reactions.
3. How is this information explained by our modern understanding of photosynthesis?
(a) Chlorophyll contains a Mg
2
ion, which is known to be an essential cofactor for many enzymes
that catalyze phosphorylation and dephosphorylation reactions.
(b) A crude “chlorophyll protein” isolated from photosynthetic cells showed phosphorylating activity.
(c) The phosphorylating activity of the “chlorophyll protein” was inhibited by light.
(d) The levels of several different phosphorylated compounds in photosynthetic cells changed dra-
matically in response to light exposure. (Emerson and coworkers were not able to identify the
specific compounds involved.)
As it turned out, the Emerson and Rabinowitch models were both partly correct and partly
incorrect.
(e) Explain how the two models relate to our current model of photosynthesis.
In his rejection of the Emerson model, Rabinowitch went on to say: “The difficulty of the phos-
phate storage theory appears most clearly when one considers the fact that, in weak light, eight or ten
quanta of light are sufficient to reduce one molecule of carbon dioxide. If each quantum should pro-
duce one molecule of high-energy phosphate, the accumulated energy would be only 80–100 kcal per
Einstein—while photosynthesis requires at least 112 kcal per mole, and probably more, because of
losses in irreversible partial reactions” (Vol. 1, p. 228).
(f) How does Rabinowitch’s value of 8 to 10 photons per molecule of CO
2
reduced compare with the
value accepted today? You need to consult Chapter 20 for some of the information required here.
(g) How would you rebut Rabinowitch’s argument, based on our current knowledge about photosyn-
thesis?
Answer
Chapter 19 Oxidative Phosphorylation and Photophosphorylation S-237
c19OxidativePhosphorylationandPhotophosphorylation.qxd 11/30/12 7:15 PM Page S-237