etc solutions

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?
The long isoprenoid side chain makes ubiquinone very soluble in lipids and allows it
to diffuse in the semifluid membrane. This is important because ubiquinone transfers electrons
from Complexes I and II to Complex III, all of which are embedded in the inner mitochondrial
membrane.
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, c1, c, and a a3.
(a) Abundant NADH and O2, but cyanide added
(b) Abundant NADH, but O2 exhausted
(c) Abundant O2, but NADH exhausted
(d) Abundant NADH and O2
(a) Cyanide inhibits cytochrome oxidase (a a3); all carriers become reduced.
(b) In the absence of O2, no terminal electron acceptor is present; all carriers become
reduced.
(c) In the absence of NADH, no carrier can be reduced; all carriers become oxidized.
(d) These are the usual conditions for an aerobic, actively metabolizing cell; the early carriers
(e.g., Q) are somewhat reduced, while the late ones (e.g., cytochrome c) are oxidized.
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.
(a) The inhibition of NADH dehydrogenase by rotenone decreases the rate of electron flow
through the respiratory chain, which in turn decreases the rate of ATP production. If this
reduced rate is unable to meet its ATP requirements, the organism dies.
(b) Antimycin A strongly inhibits the oxidation of reduced Q in the respiratory chain, severely
limiting the rate of electron transfer and ATP production.
(c) Electrons flow into the system at Complex I from the NAD-linked reactions and at
Complex II from succinate and fatty acyl-CoA through FAD (see Figs. 19-8 and 19-16).
Antimycin A inhibits electron flow (through Q) from all these sources, whereas rotenone
inhibits flow only through Complex I. Thus, antimycin A is a more potent poison.
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
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.
At relatively low levels of an uncoupling agent, P/O ratios drop somewhat, but the cell
can compensate for this by increasing the rate of electron flow; ATP levels can be kept
relatively normal. At high levels of uncoupler, P/O ratios approach zero and the cell
cannot maintain ATP levels.
When the antibiotic valinomycin is added to actively respiring mitochondria, several things happen: the yield of ATP decreases, the rate of O2
consumption increases, heat is released, and the pH gradient across the inner mitochondrial membrane
increases. Does valinomycin act as an uncoupler or an inhibitor of oxidative phosphorylation?
The observed effects are consistent with the action of an uncoupler—that is, an
agent that causes the free energy released in electron transfer to appear as heat rather than
in ATP. In respiring mitochondria, H ions are translocated out of the matrix during electron
transfer, creating a proton gradient and an electrical potential across the membrane. A significant
portion of the free energy used to synthesize ATP originates from this electric potential.
Valinomycin combines with K ions to form a complex that passes through the inner mitochondrial
membrane. So, as a proton is translocated out by electron transfer, a K ion moves
in, and the potential across the membrane is lost. This reduces the yield of ATP per mole of
protons flowing through ATP synthase (FoF1). In other words, electron transfer and phosphorylation
become uncoupled. In response to the decreased efficiency of ATP synthesis, the rate
of electron transfer increases markedly. This results in an increase in the H gradient, in oxygen
consumption, and in the amount of heat released.
When DCCD is added to a suspension of tightly coupled, actively respiring mitochondria, the rate of electron transfer (measured by O2 consumption)
and the rate of ATP production dramatically decrease

What process in electron transfer or oxidative phosphorylation is affected by DCCD?

DCCD (like oligomycin) inhibits ATP synthesis. In tightly coupled mitochondria, this inhibition leads to inhibition
of electron transfer also
DCCD – If a solution of 2,4-dinitrophenol is
now added to the preparation, O2 consumption returns to normal but ATP production remains inhibited

Why does DCCD affect the O2 consumption of mitochondria? Explain the effect of 2,4-dinitrophenol
on the inhibited mitochondrial preparation.

A decrease in electron transfer causes a decrease in O2 consumption. 2,4-Dinitrophenol
uncouples electron transfer from ATP synthesis, allowing respiration to increase. No ATP
is synthesized and the P/O ratio decreases.
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?
Malate dehydrogenase catalyzes the conversion of malate to oxaloacetate in the citric acid cycle, which takes place in the mitochondrion, and also plays a key role in the transport of reducing equivalents across the inner mitochondrial membrane via the malate-aspartateshuttle
The transport system that conveys malate and a-ketoglutarate across the inner mitochondrial membrane is inhibited by nbutylmalonate. Suppose n-butylmalonate is added to an aerobic suspension of kidney cells using glucose exclusively as fuel.

NADH produced in the cytosol cannot cross the inner mitochondrial membrane, but
must be oxidized if glycolysis is to continue. Reducing equivalents from NADH enter the mitochondrion
by way of the malate-aspartate shuttle. NADH reduces oxaloacetate to form malate
and NAD, and the malate is transported into the mitochondrion. Cytosolic oxidation of glucose
can continue, and the malate is converted back to oxaloacetate and NADH in the mitochondrion

Predict the effect of this inhibitor on (a) glycolysis, (b) oxygen consumption,
(c) lactate formation, and (d) ATP synthesis.

(a) If n-butylmalonate, an inhibitor of the malate-a-ketoglutarate transporter, is added to
cells, NADH accumulates in the cytosol. This forces glycolysis to operate anaerobically,
with reoxidation of NADH in the lactate dehydrogenase reaction.
(b) Because reducing equivalents from the oxidation reactions of glycolysis do not enter the
mitochondrion, oxygen consumption slows and eventually ceases.
(c) The end product of anaerobic glycolysis, lactate, accumulates.
(d) ATP is not formed aerobically because the cells have converted to anaerobic glycolysis.
Overall, ATP synthesis decreases drastically, to 2 ATP per glucose molecule.
When O2 is added to an anaerobic suspension of cells consuming glucose at a high rate, the rate of glucose consumption declines greatly as the O2 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 O2 is added?
(b) Why does the presence of O2 decrease the rate of glucose consumption?
(c) How does the onset of O2 consumption slow down the rate of glucose consumption? Explain in
terms of specific enzymes.
(a) Oxygen allows the tissue to convert from lactic acid fermentation to respiratory electron
transfer and oxidative phosphorylation as the mechanism for NADH oxidation.
(b) Cells produce much more ATP per glucose molecule oxidized aerobically, so less glucose
is needed.
(c) As [ATP] rises, phosphofructokinase-1 is inhibited, thus slowing the rate of glucose entry
into the glycolytic pathway.
Why does the absence of cytochrome oxidase eliminate the Pasteur effect?
The absence of cytochrome oxidase prevents these mutants from oxidizing the products
of fermentation (ethanol, acetate, lactate, or glycerol) via the normal respiratory route.
These mutants do not have a working citric acid cycle because they cannot reoxidize NADH
through the O2-dependent electron-transfer chain. Thus, catabolism of glucose stops at the
ethanol stage, even in the presence of oxygen. The ability to carry out these fermentations in
the presence of oxygen is a major practical advantage because completely anaerobic conditions
are difficult to maintain. The Pasteur effect—the decrease in glucose consumption that
occurs when oxygen is introduced—is not observed in the absence of an active citric acid
cycle and electron-transfer chain.
heteroplasmy
What term describes the presence of both normal and mutated mitochondrial DNA (mtDNA), resulting in variable expression in mitochondrial inherited diseases?
Although both pyruvate dehydrogenase and glyceraldehyde
3-phosphate dehydrogenase use NAD as their electron acceptor, the two enzymes do not compete
for the same cellular NAD pool. Why?
Pyruvate dehydrogenase is located in the mitochondrion, and glyceraldehyde
3-phosphate dehydrogenase in the cytosol. Because the mitochondrial and cytosolic pools of
NAD are separated by the inner mitochondrial membrane, the enzymes do not compete for
the same NAD pool. However, reducing equivalents are transferred from one nicotinamide
coenzyme pool to the other via shuttle mechanism
transmembrane movement of reducing equivalents
The malate-aspartate shuttle transfers electrons and protons from the cytoplasm into
the mitochondrion. Neither NAD nor NADH passes through the inner membrane, thus the labeled
NAD moiety of [7-14C]NADH remains in the cytosol. The 3
H on [4-3
H]NADH enters the
mitochondrion via the malate-aspartate shuttle (see Fig. 19-29). In the cytosol, [4-3
H]NADH
transfers its 3
H to oxaloacetate to form [3
H]malate, which enters the mitochondrion via the
malate- -ketoglutarate transporter, then donates the 3
H to NAD to form [4-3
H]NADH in the
matrix.
NADH
(1) electron donor and reducing agent
FMN
(1) electron acceptor and oxidizing agent
FMNH2
(2) electron donor and reducing agent
Fe3+
(2) electron acceptor and oxidizing agent
Fe2+
(3) electron donor and reducing agent
Q
(3) electron acceptor and oxidizing agent
adenine nucleotide translocase
The effect of replacing ATP4- with ADP3- in the matrix is the net efflux of one negative charge, which is favored by the charge difference across the inner membrane (outside positive)
an antiporter that brings ATP out of the matrix into the ims and ADP into the matrix from the ims
phosphate translocase
-is a symporter that allows a H+ to flow down its concentration gradient and simultaneously brings a phosphate group along into the matrix; is specific for H2PO4-. There is no net flow of charge during symport of H2PO4- and H+, but the relatively low proton concentration in the matrix favors the inward movement of H+. Thus the proton-motive force is responsible both for providing the energy for ATP synthesis and for transporting substrates (ADP and Pi) into and product (ATP) out of the mitochondrial matrix.
Glycerol 3-phosphate shuttle
This alternative means of moving reducing equivalents from the cytosol to the mitochondrial matrix operates in skeletal muscle and the brain. In the cytosol, dihydroxyacetone phosphate accepts two reducing equivalents from NADH in a reaction catalyzed by cytosolic glycerol 3-phosphate dehydrogenase. An isozyme of glycerol 3-phosphate dehydrogenase bound to the outer face of the inner membrane then transfers two reducing equivalents from glycerol 3-phosphate in the intermembrane space to ubiquinone. Note that this shuttle does not involve membrane transport systems.
FoF1 complex
protein complex in the mitochondrial inner membrane and the bacterial plasma membrane that consists of the F1 complex bound to the Fo complex; the flow of protons through the Fo component leads to the synthesis of ATP by the F1 component.
High [ATP] or low [ADP] and [AMP]
produces low rates of glycolysis, pyruvate oxidation, acetate oxidation via the citric acid cycle, and oxidative phosphorylation. All four pathways are accelerated when the use of ATP and the formation of ADP, AMP, and Pi increase.
citrate
inhibits glycolysis, supplements the action of the adenine nucleotide system
increased levels of NADH and acetyl-CoA
also inhibit the oxidation of pyruvate to acetyl-CoA
a high [NADH]/[NAD+] ratio
inhibits the dehydrogenase reactions of TCA
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 uncouplers?
As amounts of an uncoupler increase, the P/O ratio decreases and the body struggles to
make sufficient ATP by oxidizing more fuel. The heat produced by this increased rate of
oxidation raises the body temperature
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?
Increased activity of the respiratory chain in the presence of an uncoupler requires the
degradation of additional energy stores (glycogen and fat). By oxidizing more fuel in an
attempt to produce the same amount of ATP, the organism loses weight
Single nucleotide changes in the gene for succinate
dehydrogenase (Complex II) are associated with midgut carcinoid tumors
Defects in Complex II result in increased production of ROS, damage to DNA, and
mutations that lead to unregulated cell division (cancer)
cancer cells are in an altered metabolic state; mutations in mitochondrial DNA encoded genes can contribute to the development of cancer. It is possible that such mutations provide metabolic adaptivity to the cancer cell
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