BC chap 21
Electromyogram
 Measures electrical potential of muscle cells at rest and while contracting
 10-20 measurements taken at various places
 Under normal conditions, muscles at rest will have minimal electrical activity
 Abnormal electromyograms suggest underlying pathology interfering with membrane polarizationrepolarization as nerve cells instruct muscle cells to contract
Clinical Notes
 Follicular-type non-Hodgkin lymphoma – treated with doxorubicin
o Although doxorubicin is highly effective anticancer agent against wide variety of tumors, its clinical use
limited by specific, cumulative, dose-dependent cardiotoxicity
o Impairment of mitochondrial function may play major role in toxicity
o Doxorubicin binds to cardiolipin (lipid component of inner membrane of mitochondria), where it might
directly affect components of oxidative phosphorylation
o Doxorubicin inhibits succinate oxidation, inactivates cytochrome oxidase, interacts with CoQ, adversely
affects ino pumps, and inhibits ATP synthase, resulting in decreased ATP levels and mildly swollen
mitochondria; decreases ability of mitochondria to sequester Ca2+ and increases free radicals, leading to
damage of mitochondrial membrane
o Also might affect heart function indirectly through other mechanisms
Oxidative Phosphorylation
 Generation of ATP from oxidative phosphorylation requires electron donor (NADH or FAD(2H)), electron
acceptor (O2), intact inner mitochondrial membrane impermeable to protons, all components of electron
transport chain, and ATP synthase; regulated by rate of ATP use
 Most cells dependent on oxidative phosphorylation for ATP homeostasis
o During ischemia, inability to generate energy from electron-transport chain results in increased
permeability of membrane and mitochondrial swelling
o Mitochondrial swelling is key element in pathogenesis of irreversible cell injury leading to cell lysis and
death (necrosis)
Overview of Oxidative Phosphorylation
 Understanding of oxidative phosphorylation based on chemiosmotic hypothesis (energy for ATP synthesis
provided by electrochemical gradient across inner mitochondrial membrane)
o Electrochemical gradient generated by components of electron-transport chain, which pumps protons
across inner mitochondrial membrane as they sequentially accept and donate electrons; final acceptor is
O2, which is reduced to H2O
 Electrons donated by NADH or FAD(2H) passed sequentially through series of electron carriers embedded in
inner mitochondrial membrane; each component reduced as it accepts electron and then oxidized as it passes
electrons to next member of chain
o From NADH, electrons transferred sequentially through NADH:CoQ oxidoreductase (complex I),
coenzyme Q (CoQ), cytochrome b-c1 complex (complex III), cytochrome c, and finally cyrochrome c
oxidase (complex IV)
o NADH:CoQ oxidoreductase, cytochrome b-c1 complex, and cytochrome c oxidase each multi-subunit
protein complexes that span inner mitochondrial membrane
o CoQ is lipid-soluble quinone that isn’t protein bound and free to diffuse in lipid membrane; transports
electrons from complex I to complex III and is intrinsic part of proton pump for each complex
o Cytochrome c is small protein in intermembrane space that transfers electrons from b-c1 complex to
cytochrome oxidase
o Terminal complex (cytochrome c oxidase) contains binding site for O2; as O2 accepts electrons from
chain, it is reduced to H2O
 At each of the 3 large membrane-spanning complexes in the chain, electron transfer accompanied by proton
pumping across membrane
o Energy drop of about 16 kcal as electrons pass through each complex, which provides energy required to
move protons against the concentration gradient
o In actively respiring mitochondria, intermembrane space and cytosol is 0.75 pH unit lower than matrix
o
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Transmembrane movement of protons generates electrochemical gradient with 2 components:
membrane potential (external face of membrane + relative to matrix side) and proton gradient
(intermembrane space has higher proton concentration and is therefore more acidic than matrix)
 Gradient sometimes called proton motive force because it is the energy that pushes protons to
reenter matrix to equilibrate on both sides of membrane
 Protons attracted to more negative matrix side of membrane, where pH is more alkaline
ATP synthase (F0F1 ATPase) – enzyme that generates ATP; multi-subunit enzyme that contains inner membrane
portion (F0) and stalk with headpiece (F1) that projects into matrix
o 12 C-subunits in membrane form rotor that is attached to central asymmetric shaft composed of ε and
γ-subunits
o Headpiece composed of 3 αβ-subunit pairs; each β-subunit contains catalytic site for ATP synthesis
o Headpiece held stationary by δ-subunit attached to long b-subunit connected to a-subunit in membrane
o Influx of protons through proton channel turns rotor
o Proton channel formed by c-subunit on one side and a-subunit on other
 Although channel continuous, it has 2 offset portions
 One portion open directly to intermembrane space
 One portion open directly to matrix
 Each c-subunit contains glutamyl carboxyl group that extends into protein channel; because
carboxyl group accepts proton from intermembrane space, c-subunit rotates into hydrophobic
lipid membrane
 Rotation exposes different protein-containing c-subunit to portion of channel open directly to
matrix side
 Because matrix has lower proton concentration, glutamyl carboxylic acid group releases proton
into matrix portion of channel
 Rotation completed by attraction between negatively charged glutamyl residue and positively
charged arginyl group on a-subunit
o As asymmetric shaft rotates to new position, it forms different binding associations with αβ-subunits;
new position of shaft alters conformation of one β-subunit so it releases molecule of ATP and another
subunit spontaneously catalyzes synthesis of ATP from inorganic phosphate, one proton, and ADP
 Energy from electrochemical gradient used to change conformation of ATP synthase subunits so
newly synthesized ATP released
 Takes 12 protons to complete one turn of rotor and synthesize 3 ATPs
Oxidation-Reduction Components of Electron-Transport Chain
 Redox components of chain include flavin mononucleotide (FMN), iron-sulfur (Fe-S) centers, CoQ, and Fe in
cytochromes b, c1, c, a, and a3; Cu also component of cytochromes a and a3
o With exception of CoQ, all electron acceptors tightly bound to protein subunits of carriers
o FMN (like FAD) synthesized from vitamin riboflavin
o Reduction potential of each complex at lower energy level than previous complex, so energy released as
electrons pass through each complex
 Energy released used to move protons against their concentration gradient, so they become
concentrated on cytosolic side of inner membrane
 NADH:CoQ oxidoreductase (also called NADH dehydrogenase) is 42-subunit complex that contains binding site
for NADH, several FMN, Fe-S center-binding proteins, and binding sites for CoQ
o
FMN accepts 2 electrons from NADH and passes single electrons to Fe-S centers, which are able to
delocalize single electrons into large orbitals
o Fe-S centers transfer electrons to and from CoQ
o Fe-S centers also present in other enzyme systems, such as proteins in cytochrome b-c1 complex, which
transfer electrons to CoQ, and in aconitase in TCA cycle
 Succinate dehydrogenase and flavoproteins in inner mitochondrial membrane pass electrons to CoQ
o Succinate dehydrogenase part of TCA cycle and component of complex II of electron-transport chain
o ETF-CoQ oxidoreductase accepts e- from ETF (electron-transferring flavoprotein), which acquires them
from fatty acid oxidation and other pathways
o Both flavoproteins have Fe-S centers
o Glycerol-3-phosphate dehydrogenase is flavoprotein that’s part of shuttle for reoxidizing cytosolic NADH
o Free-energy drop in electron transfer between NADH and CoQ able to support movement of 4 proteins
o FAD in succinate dehydrogenase (as well as ETF-CoQ oxidoreductase and glycerol-3-phosphate
dehydrogenase) at roughly same redox potential as CoQ, and no energy released as they transfer
electrons to CoQ
o Complex II doesn’t span membrane and consequently doesn’t have proton-pumping mechanism
 CoQ – only component of electron-transport chain not protein bound; able to diffuse through lipids of inner
mitochondrial membrane
o When oxidized quinone form accepts single electron, it forms free radical; transfer of single electrons
makes it major site for generation of toxic oxygen free radicals in body
o Semiquinone can accept second electron and 2 protons from matrix side of membrane to form fully
reduced quinone
o Mobility of CoQ in membrane, ability to accept 1 or 2 electrons, and ability to accept and donate
protons enable it to participate in proton pumps for both complexes I and III as it shuttles electrons
between them
o CoQ also called ubiquinone because quinones with similar structures found in all plants and animals
 Cytochromes – each is protein that contains bound heme; because of differences in protein component of
cytochromes and small differences in heme structure, each heme has different reduction potential
o Cytochromes of b-c1 complex have higher energy level than those of cytochrome oxidase (a and a3); thus
energy released by electron transfer between complexes III and IV
o Iron atoms in cytochromes are in Fe3+ state; as they accept electron, they are reduced to Fe2+
o As they are reoxidized to Fe3+, electrons pass to next component of electron-transport chain
o Although iron deficiency anemia characterized by decreased levels of Hgb and other Fe-containing
proteins in blood, Fe-containing cytochromes and Fe-S centers of electron-transport chain in tissues
such as skeletal muscle affected as rapidly; fatigue in iron deficiency anemia results in part from lack of
electron transport for ATP production
 Last cytochrome complex is cytochrome oxidase, which passes electrons from cytochrome c to O2
o Contains cytochromes a and a3 and oxygen-binding site
o Whole O2 molecule must accept 4 e- to be reduced to 2 H2O molecules
o Bound Cu+ in cytochrome oxidase complex facilitate collection of 4 e- and reduction of O2
o Cytochrome oxidase has much lower Km for O2 than myoglobin (heme-containing intracellular oxygen
carrier) or Hgb, so O2 pulled from erythrocyte to myoglobin and from myoglobin to cytochrome oxidase
 Normally, protein structures binding heme either protect iron from oxidation (such as globin proteins) or allow
oxidation to occur (such as happens in cytochromes)
o Hemoglobin M (rare Hgb variant) has tyrosine substituted for histidine at position F8 in normal Hgb A;
tyrosine stabiliaes Fe3+ form of heme, and these subunits can’t bind O2; lethal condition if homozygous
Pumping of Protons
 Energy from redox reactions of electron-transport chain used to transport protons from matrix to
intermembrane space
 Proton pumping generally facilitated by vectorial arrangement of membrane-spanning complexes; structure
allows them to pick up electrons and protons on one side of membrane and release protons on other side of
membrane as they transfer electron to next component of chain
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Q cycle involves double cycle of CoQ reduction and oxidation; CoQ accepts 2 protons at matrix side together
with 2 electrons, then releases protons into intermembrane space while donating one electron back to another
component of cytochrome b-c1 complex and one to cytochrome c
 Mechanism for pumping protons at NADH:CoQ oxidoreductase complex involves Q cycle in which Fe-S centers
and FMN participate
 Transmembrane proton movement at cytochrome c oxidase involves direct transport of proton through series of
bound water molecules or amino acid side chains in protein (protein wire)
 Significance of direct link between electron transfer and proton movement is one can’t occur without the other
o When protons not being used for ATP synthesis, proton gradient and membrane potential build up
o Proton back pressure controls rate of proton pumping, which controls electron transport and O2
consumption
Energy Yield from Electron-Transport Chain
 Overall free-energy release from oxidation of NADH by O2 and Fad(2H) has ΔG0 so negative that chain is never
reversible; can never synthesize O2 from H2O; drives NADH and FAD(2H) formation from pathways of fuel
oxidation, such as TCA cycle and glycolysis, to completion
 Overall each NADH donates 2 electrons (reduction of ½ O2 molecule)
 4 protons pumped at complex I, 4 protons at complex III, and 2 protons at complex IV
o With 4 protons translocated for each ATP synthesized, estimated 2.5 ATPs formed for each NADH
oxidized
o 1.5 ATPs formed for each FAD(2H)-containing flavoproteins that donate electrons to CoQ
o Only 30% of energy available from NADH and FAD(2H) oxidation by O2 used for ATP synthesis; some of
remaining energy in electrochemical potential used for transport of anions and Ca2+ into mitochondrion
o Remainder of energy released as heat
 Electron-transport chain is major source of heat
Respiratory Chain Inhibition and Sequential Transfer
 In cell, electron flow in electron-transport chain must be sequential from NADH or flavoprotein all the way to O2
to generate ATP; in absence of O2, no ATP generated from oxidative phosphorylation because electrons back up
 Limited availability of O2 (as in ischemia) to act as electron acceptor will decrease proton pumping and
generation of electrochemical potential gradient across inner mitochondrial membrane of ischemic cells; rate of
ATP generation in these specific areas will decrease, triggering events that lead to irreversible cell injury
 In absence of O2, even complex I can’t pump protons to generate electrochemical gradient because every
molecule of CoQ already has electrons that it can’t pass down the chain without O2 to accept them at the end
 Action of respiratory chain inhibitor cyanide, which binds to cytochrome oxidase, similar to that of anoxia;
prevents proton pumping by all 3 complexes
o Complete inhibition of b-c1 complex prevents pumping at cytochrome oxidase because there is no donor
of electrons; prevents pumping at complex I because there is no electron acceptor
 Mitochondrial respiration and energy production cease, and cell death rapidly occurs
 CNS is primary target for cyanide toxicity
o Intravenous nitroprusside rapidly lowers elevated blood pressure through direct vasodilating action;
during prolonged infusions of 24-48 hours, nitroprusside converted to cyanide (inhibitor of cytochrome c
oxidase complex – binds to Fe3+ in heme of cytochrome aa3 component)
 Because small amounts of cyanide detoxified in liver by conversion to thiocyanate, which is
excreted in urine, conversion of nitroprusside to cyanide can be monitored by following blood
thiocyanate levels
 Complete inhibition of any one complex inhibits proton pumping at all complexes
 Partial inhibition of proton pumping can occur when only fraction of molecules of complex contains bound
inhibitor; results in partial decrease of maximal rate of ATP synthesis
Oxidative Phosphorylation Diseases
 Clinical diseases involving components of oxidative phosphorylation (OXPHOS diseases) among most commonly
encountered degenerative diseases
o Clinical pathology may be caused by gene mutations in either mtDNA or nuclear DNA (nDNA) that
encodes proteins required for normal oxidative phosphorylation
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OXPHOS responsible for producing most of ATP cells require; genes responsible for polypeptides that comprise
OXPHOS complexes within mitochondria located in either nDNA or mtDNA
o Broad spectrum of disorders (OXPHOS diseases) may results from genetic mutations or nongenetic
alterations (spontaneous mutations) in either nDNA or mtDNA
o Increasingly, such changes responsible for some aspects of common disorders (Parkinson disease,
dilated and hypertrophic cardiomyopathies, DM, Alzheimer’s, depressive disorders, and host of less
well-known clinical entities)
mtDNA – small, double-stranded, circular DNA; encodes 13 subunits of complexes involved in oxidative
phosphorylation (7 subunits of complex I, 1 subunit of complex III, 3 subunits of complex IV, and 2 subunits of F0
portion of ATP-synthase complex)
o Also encodes necessary components for translation of its mRNA (large and small rRNA and 22 tRNAs)
o Genetics of mutations in mtDNA defined by maternal inheritance, replicative segregation, threshold
expression, high mtDNA mutation rate, and accumulation of somatic mutations with age
 Usually, some mitochondria present that have mutant mtDNA and some have normal DNA; as
cells divide, mitochondria replicate by fission, but various amounts of mitochondria with mutant
and wild-type DNA distributed to each daughter cell (replicative segregation)
 Any cell can have mixture of mitochondria, each with mutant or wild-type mtDNAs
(heteroplasmy)
 Mitotic and meiotic segregation of heteroplasmic mtDNA mutation results in variable
oxidative phosphorylation deficiencies between patients with same mutation and even
among patient’s own tissues
o Disease pathology usually becomes worse with age because small amount of normal mitochondria
might confer normal function and exercise capacity while patient is young, but as patient ages,
spontaneous mutations in mtDNA accumulate from generation of free radicals in mitochondria
 Mutations frequently become permanent, partly because mtDNA doesn’t have access to same
repair mechanisms available for nDNA (high mutation rate)
 Even in normal individuals, somatic mutations result in decline of oxidative phosphorylation
capacity with age (accumulation of somatic mutations with age)
 At some stage, ATP-generating capacity of tissue falls below tissue-specific threshold for normal
function (threshold expression)
o In general, symptoms of defects appear in one or more tissues with highest ATP demands: nervous
tissue, heart, skeletal muscle, and kidney
o Myoclonic epileptic ragged red fiber disease (MERRF) – spontaneous muscle jerking (myoclonus) that
starts in midteens and progresses over 10 years to include debilitating myoclonus, neurosensory hearing
loss, dementia, hypoventilation, and mild cardiomyopathy
 Energy metabolism affected in CNS, heart, and skeletal muscle, resulting in lactic acidosis
 Maternally inherited
 Affected tissues are those with highest ATP requirements
 Most cases caused by point mutation in mtRNALys
 Mitochondria (obtained by muscle biopsy) enlarged and show abnormal patterns of cristae
 Muscle tissue shows ragged red fibers
Effect of inhibition of electron transport is impaired oxidation of pyruvate, fatty acids, and other fuels
o In many cases, inhibition of mitochondrial electron transport results in higher than normal levels of
lactate and pyruvate in blood and increased lactate/pyruvate ratio
o NADH oxidation requires complete transfer of electrons from NADH to O2, and defect anywhere along
chain will result in accumulation of NADH and decrease of NAD+
o Increase in NADH/NAD+ inhibits pyruvate dehydrogenase and causes accumulation of pyruvate;
increases conversion of pyruvate to lactate, and elevated levels of lactate appear in blood
o Large number of genetic defects of proteins in respiratory chain complexes classified together as
congenital lactic acidosis
Genetic mutations for mitochondrial proteins encoded by nDNA; nDNA encodes rest of subunits of oxidative
phosphorylation complexes, as well as adenine nucleotide translocase (ANT) and other anion translocators
o
Coordinate regulation of expression of nDNA and mtDNA, importation of proteins into mitochondria,
assembly of complexes, and regulation of mitochondrial fission nuclear encoded
o Nuclear respiratory factors (NRF-1 and NRF-2) – nuclear transcription factors that bind to and activate
promoter regions of nuclear genes that encode subunits of respiratory chain complexes, including
cytochrome c
 Also activate transcription of nuclear gene for mitochondrial transcription factor-A (mTF-A);
protein product of this gene translocates into mitochondrial matrix, where it stimulates
transcription and replication of mitochondrial genome
o nDNA mutations differ from mtDNA mutations because nDNA mutations usually autosomal recessive
 Mutations uniformly distributed to daughter cells and therefore expressed in all tissues that
contain allele for particular tissue-specific isoform
 Phenotypic expression most apparent in tissues with high ATP requirements
Coupling of Electron Transport and ATP Synthesis
 Electrochemical gradient couples rate of electron-transport chain to rate of ATP synthesis
 Because electron flow requires proton pumping, electron flow can’t occur faster than protons are used for ATP
synthesis (coupled oxidative phosphorylation) or returned to matrix by mechanism that short-circuits ATP
synthesis pore (uncoupling)
 As ATP chemical bond energy used by energy-requiring reactions, ADP and Pi concentrations increase
o The more ADP present to bind to ATP synthase, the greater the proton flow through ATP synthase pore
from intermembrane space to matrix
o As ADP levels rise, proton influx increases, and electrochemical gradient decreases
o Proton pumps of electron-transport chain respond with increased proton pumping and electron flow to
maintain electrochemical gradient; result is increased O2 consumption
 Increased oxidation of NADH in electron-transport chain and increased concentration of ADP stimulate
pathways of fuel oxidation, such as TCA cycle, to supply more NADH and FAD(2H) to electron-transport chain
o During exercise, we use more ATP for muscle contraction, consume more oxygen, oxidize more fuel
(burn more calories), and generate more heat from electron-transport chain
 Shivering results from muscular contraction, which increases rate of ATP hydrolysis; as
consequence of proton entry for ATP synthesis, electron-transport chain stimulated; oxygen
consumption increases, as does amount of energy lost as heat by electron-transport chain
o If we rest, and rate of ATP use decreases, proton influx decreases, electrochemical gradient increases,
and proton back pressure decreases rate of electron-transport chain
 NADH and FAD(2H) can’t be oxidized as rapidly in electron-transport chain, and consequently,
their buildup inhibits enzymes that generate them
o System poised to maintain very high levels of ATP at all times; in most tissues, rate of ATP use nearly
constant over time
 Even under extreme variation (as in skeletal muscle), ATP concentration decreases by only 20%
because it is so rapidly regenerated
 In heart, Ca2+ activation of TCA cycle enzymes provides extra push to NADH generation so that
neither ATP nor NADH levels fall as ATP demand increased
o Electron-transport chain has very high capacity and can respond very rapidly to any increase in ATP use
 When protons leak back into matrix without going through ATP synthase pore, they dissipate electrochemical
gradient across membrane without generating ATP (uncoupling oxidative phosphorylation)
o Occurs with chemical compounds (uncouplers) and physiologically with uncoupling proteins that form
proton conductance channels through membrane
o Uncoupling of oxidative phosphorylation results in increased oxygen consumption and heat production
as electron flow and proton pumping attempt to maintain electrochemical gradient
o Chemical uncouplers (proton ionophores) are lipid-soluble compounds that rapidly transport protons
from cytosolic to matrix side of inner mitochondrial membrane
 Because proton concentration is higher in intermembrane space than in matrix, uncouplers pick
up protons from intermembrane space
 Lipid solubility enables them to diffuse through inner mitochondrial membrane while carrying
protons and to release protons on matrix side
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Mitochondria unable to synthesize ATP, and eventually mitochondrial integrity and function lost
Zidovudine (azidothymidine or AZT) – used to treat AIDS and can act as inhibitor of mtDNA
polymerase (polymerase γ); can rarely cause varying degrees of mtDNA depletion in different
tissues, including skeletal muscle; depletion may cause severe mitochondrial myopathy,
including ragged red fiber accumulation in skeletal muscle cells associated with ultrastructural
abnormalities in mitochondria
 Salicylate (degradation product of aspirin) is lipid-soluble and has dissociable proton; in high
concentrations, salicylate able to partially uncouple mitochondria
 Decline of ATP concentration in cell and consequent increase of AMP in cytosol
stimulates glycolysis
 Overstimulation of glycolytic pathway results in increased levels of lactic acid in blood
and metabolic acidosis
o Uncoupling proteins (UCPs) form channels through inner mitochondrial membrane that are able to
conduct protons from intermembrane space to matrix, thereby short-circuiting ATP synthase
 UCP1 (thermogenin) associated with heat production in brown adipose tissue
 Major function of brown adipose tissue is non-shivering thermogenesis; major function
of white adipose tissue is storage of triacylglycerols in white lipid droplets
 Brown color arises from large number of mitochondria that participate
 Infants have little voluntary control over environment and may kick blankets off at
night; have brown fat deposits along neck, breastplate, between scapulae, and around
kidneys to protect them from cold
 In response to cold, SNS nerve endings release norepinephrine, which activates lipase in
brown adipose tissue that releases fatty acids from triacylglycerols
 Fatty acids serve as fuel for tissue (i.e., are oxidized to generate electrochemical
potential gradient and ATP) and participate directly in proton conductance channel by
activating UCP1 along with reduced CoQ
 When UCP1 activated by fatty acids, it transports protons from cytosolic side of inner
mitochondrial membrane back into mitochondrial matrix without ATP generation;
partially uncouples oxidative phosphorylation and generates additional heat
 Uncoupling proteins exist as family of proteins; highly regulated proteins that, when
activated, increase amount of energy from fuel oxidation that is being released as heat
 UCP1 (thermogenin) expressed in brown adipose tissue
 UCP2 found in most cells
 UCP3 found principally in skeletal muscle; acts as transport protein to remove fatty acid
anions and lipid peroxides from mitochondria, thereby reducing risk of forming oxygen
free radicals and thus decreasing occurrence of mitochondrial and cell injury
 UCP4 and UCP5 found in nervous system
o Low level of proton leak across inner mitochondrial membrane occurs in mitochondria all the time;
mitochondria normally partially uncoupled
 More than 20% of resting metabolic rate is energy expended to maintain electrochemical
gradient dissipated by basal proton leak (global proton leak)
 Some of proton leak results from permeability of membrane associated with proteins
embedded in lipid bilayer
 Unknown amount may result from uncoupling proteins
Transport through Inner and Outer Mitochondrial Membranes
 Most of newly synthesized ATP released into mitochondrial matrix must be transported out of mitochondria
where it is used for energy-requiring processes such as active ion transport, muscle contraction, or biosynthetic
reactions; ADP, phosphate, pyruvate, and other metabolites must be transported into matrix as well; requires
transport of compounds through both inner and outer mitochondrial membranes
 Inner mitochondrial membrane forms tight permeability barrier to all polar molecules (including ATP, ADP, P i,
anions such as pyruvate, and cations); process of oxidative phosphorylation depends on rapid and continuous
transport of many molecules across inner mitochondrial membrane
o
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Ions and other polar molecules transported across inner mitochondrial membrane by specific protein
translocases that nearly balance charge during transport process
o Most of exchange transport is form of active transport that generally uses energy from electrochemical
potential gradient (either membrane potential or proton gradient)
o ATP-ADP translocase (ANT) transports ATP formed in mitochondrial matrix to intermembrane space in
specific 1:1 exchange for ADP produced from energy-requiring reactions outside mitochondria
 Because ATP contains 4 negative charges and ADP contains only 3, exchange promoted by
electrochemical potential gradient because net effect is transport of one negative charge from
matrix to cytosol
 Similar antiports exist for most metabolic anions
o Inorganic phosphate and pyruvate transported into mitochondrial matrix on specific transporters
(symports) together with proton
o Specific transport protein for Ca2+ uptake (Ca2+ uniporter) driven by electrochemical potential gradient,
which is negatively charged on matrix side of membrane
o Other transporters include dicarboxylate transporter (phosphate-malate exchange), tricarboxylate
transporter (citrate-malate exchange), aspartate-glutamate transporter, and malate-α-ketoglutarate
transporter
Outer mitochondrial membrane is permeable to compounds with MW up to 6 kDa because it contains large
nonspecific pores (voltage-dependent anion channels (VDACs) formed by mitochondrial porins)
o VDACs composed of porin homodimers that form β-barrel with relatively large nonspecific water-filled
pore through center; channels open at low transmembrane potential, with preference for anions
(pyruvate, citrate, adenine nucleotides)
 Facilitate translocation of anions between intermembrane space and cytosol
o Several cytosolic kinases (such as hexokinase that initiates glycolysis) bind to cytosolic side of channel
where they have ready access to newly synthesized ATP
Mitochondrial permeability transition involves opening of large nonspecific pore (MPTP or mitochondrial
permeability transition pore) through inner mitochondrial membrane and outer membranes at sites where they
form junction
o Basic components of pore are ANT, VDAC, and cyclophilin D (CD; a cis-trans isomerase for proline
peptide bond)
 Normally ANT closed pore that functions specifically in 1:1 exchange of matrix ATP for ADP in
intermembrane space; increased mitochondrial matrix Ca2+, excess phosphate, or reactive
oxygen species (form oxygen or oxygen-nitrogen radicals) can activate opening of pore
 ATP on cytosolic side of pore (and possibly acidic pH) and membrane potential across inner
membrane protect against pore opening
o Opening of MPTP triggered by ischemia (hypoxia), which results in temporary lack of O2 for maintaining
proton gradient and ATP synthesis
 When proton gradient not being generated by electron-transport chain, ATP synthase runs
backward and hydrolyzes ATP in attempt to restore gradient, rapidly depleting cellular ATP
 As ATP hydrolyzed to ADP, ADP converted to adenine, and nucleotide pool no longer able to
protect against pore opening; can lead to downward spiral of cellular events
 Lack of ATP for maintaining low intracellular Ca2+ can contribute to pore opening
o When MPTP opens, protons flood in and maintaining proton gradient becomes impossible
 Anions and cations enter matrix, mitochondrial swelling ensues, and mitochondria become
irreversibly damaged; results in cell lysis and death by necrosis
As ATP hydrolyzed during muscular contraction, ADP formed; ADP must exchange into mitochondria on ATP-ADP
translocase to be converted back to ATP; inhibition of ATP-ADP translocase results in rapid depletion of cytosol
ATP levels and loss of cardiac contractility
Although reintroduction of oxygen to hypoxic tissue may rapidly restore capacity to generate ATP, it often
increases cell death (ischemia-reperfusion injury)
o During ischemia, stimulation of anaerobic glycolysis in cytosol generates ATP without oxygen because
glucose converted to lactic acid
o Both cytosolic ATP and lowering of pH protect against opening of MPTP
o
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Ca2+ uptake by mitochondria requires membrane potential, and matrix Ca2+ activates opening of MPTP
Depending on severity of ischemic insult, MPTP may not open, or may open and reseal, until oxygen
reintroduced; then, depending on sequence of events, reestablishment of proton gradient,
mitochondrial uptake of Ca2+, or increase of pH above 7.0 may activate MPTP before cell has recovered
o Reintroduction of O2 generates oxygen free radicals, particularly through free radical forms of CoQ in
electron-transport chain; these may open MPTP
Mitochondria and Apoptosis
 Loss of mitochondrial integrity is major route initiating apoptosis
 Intermembrane space contains procaspases 2, 3, and 9, which are proteolytic enzymes in zymogen form
o Also contains apoptosis-initiating factor (AIF) and caspase-activated DNAse (CAD)
o AIF has nuclear-targeting sequence and is transported into nucleus under appropriate conditions; once
AIF inside nucleus, it initiates chromatin condensation and degradation
o Cytochrome c (loosely bound to inner mitochondrial membrane) may also enter intermembrane space
when electrochemical potential gradient lost; release of cytochrome c and other proteins into cytosol
initiates apoptosis