Electron Transport Chain

Intermembrane space: serves to localize & concentrate enzymes (i.e. Cyt C)

Matrix: rich in enzymes & substrates involved in ATP synthesis/receives O2 & fuel delivered by blood flow/
FattyAcid degradation & TCA = primary sources of NADH & FADH2substrates to ETC
Inner Membrane (IM)
Composed of 3 lipids: Phosphatidyl-Choline (PC-3)/Phosphatidyl-Ethanolamine (PE-2)/Cardiolipin
(diphosphatidyl glycerol) (CL-1)these 3 make up most of the lipids in the IM but it also contains neutral &
other phospholipids.
Cardiolipin: Unique to inner membrane/not seen in the outer or any other membrane in cell.
IM Extremely high in proteins (65-80%) of membrane
ETC complexes I, II, III, IV are located here!
Cristae: folded to increase surface area for enzyme amount/restrict diffusion thru matrix & w/in inter-membrane
space/allows localized pH gradients (chemical & electrical) across the inner membrane
Outer Membrane (OM)
OM 20% protein
Has mito porin VDAC (Voltage-Dependent Anion Channel)makes OM permeable to molecules <6000
Daporous so has no chemical or electrical gradient

ENZYMES in diff Mito Compartments
Outer Membrane Intermembrane Space Inner Membrane Matrix
VDAC

Cytochrome C

ETC enzyme
Complexes
ATP Synthase
(Fo Subunit)
(transporters for
metabolites=AT
P, pyruvate &
citrate)

ATP synthase (F1 subunit)
TCA enzymes (Citrate
Synthase/Isocitrate
Dehydrogenase/Fumarase/M
alate Dehydrogenase
Fatty Acid Oxidation
enzymes

Know the Chemiosmotic theory
Peter Mitchell (1978) proposed that an electrochemical gradient (proton/electrical gradient) is formed across IM
by the flow of electrons thru the respiratory chain & in turn ATP molecules are synthesized by the gradient.
The inter-membrane space typically has more H+ so its pH is lower than the Matrixmatrix side more basic
by 1.4 pH units, So membrane potential across IM, (+) outside (-)inside matrix (less protons).
When protons move from cytosolic side to matrix, the free energy will be -21.8 kJ/mol (spontaneous & can
be coupled to drive other rxns
Electrical Potential Diff Voltage / Proton Concentration Diff Osmotic Pressure
Proton-motive Force (PMF):
1) Is generated by ETC
2) pH gradient & membrane potential used to drive ATP synthesis






Understand the role of the electron transport chain in the cell.
ETC: composed of 4 physically separable units (Complexes)
The oxidation/reduction rxns of Complex I, III, IV=result in pumping protons out of the matrix creating a
proton potential (electrochemical potential=EP)
The EP is then used by ATP synthase to drive phosphorylation of ADP to ATP
Proton pumping=ETC
ATP synthesis=uses Proton Motive Force
Oxidative Phosphorylation=Process for making ATPresponsible for 34 of the 36 ATP molecules formed by the
oxidation of one glucose molecule.
Cell obtains energy from foodenergy from oxidation of Fatty acids & sugars coupled to reduction of NAD &
FAD to form NADH & FADH2which are then Oxidized by mito ETCenergy from these oxidative-reductive
events is utilized to make ATP which is used by the cell for biosynthetic rxns, active transport systems & other
energy requiring processes!
1) Glycolsis in cytosol 2) Pyruvatemito matrix by Pyruvate Carrier 3) PDH complex converts PyruvateAcetyl
CoA 4) TCA takes Acetyl CoA to produce NADH & FADH2 5) ETC uses these for electrochemical potential 6)
ADP ATP via ATP Synthase
Summary: Energy from oxidation rxns converted into EP, & then the energy of EP is used for phosphorylation of
ADP to ATP= entire process is Oxidative Phosphorylation

Know the names (and alternative names) of the components of the electron transport chain: Complex I,
Complex II, Complex III and Complex IV.
ETC complexes are enzymes for oxidation/reduction of substrates
Alternate Names Sub
units
Prosthetic
Group
Oxidant/Reductant # Protons
Pumped
Inhibitors/
Diseases
I NADH Dehydrogenase
NADH-Q Oxidoreductase
NADH-Coenzyme Q Reductase
NADH: Ubiquinone
Oxidoreductase

45 FMN: 2e-
acceptor/
Hydride)
Fe-S: 1e-
carrier
Matrix: NADH
Membrane core: Q
4 Amytal
Rotenone
Myxothiazol
Piericidin A
LHON/MELAS
Leigh Syndrome
II Succinate Dehydrogenase
Succinate-Q Reductase
Complex
4 FAD
Fe-S

Matrix: Succinate
Membrane core: Q
0 Malonate
Leigh Syndrome
Certain Tumors
III Q-cytochrome c
oxidoreductase
Ubiquinone: Cyto c
Oxidoreductase
Cyto b-c1 Reductase
Cyto bc1 Complex
11 Heme bH
Heme bL
Heme c1
Fe-S
Membrane core: Q
Cytoplasm:
Cytochrome c
4 Stigmatellin (P)
Myxothiazol (P)
Antimycin A (N)
Ilicicolin (N)
Encephalomyop
athy
IV Cytochrome c Oxidase
Cytochrome Oxidase
13 Heme a
Heme a3
CuA &
CuB
Cytoplasm:
Cytochrome c
2 Cyanide/Azide
CO
Encephalomyop
athy/Myopathy

Oxidases: catalyze the removal of Hydrogen from a substrate using O2 as hydrogen acceptor
They form H20 or H2O2 as rxn product
AH2(red) + 1/2O2 A(ox) + H2O
AH2(red) + O2 A(ox) + H2O2
Dehydrogenases: transfer hydrogen from 1 substrate to another in coupled Oxidation-Reduction rxn w/o using
molecular O2 AH(red) + B(ox) A(ox) + BH(red)
**Reductase = reduct(ion)ase = oxidoreductase



Complex I (aka NADH Dehydrogenase/NADH CoQ Reductase): LARGEST
45 subunits: some in IM and others in Matrix
Active site for NADH in the inner side of IM so NADH has to be in Matrix space for access to site
Complex oxidizes NADH & reduces Q (Ubiquinone/Coenzyme) QH2 (Ubiquinol) = favorable rxn &
complex uses some of this energy to pump 4 protons out of matrix into inter-membrane space generating
EP across inner membrane.
3 Steps:
Step1: Oxidation of NADH NADHNAD + H+ (matrix) + 2e-
Step2: Reduction of Coenzyme Q Q + 2e- + 2H+ (matrix) QH2 (contains stoichiometric protons)
Step3: Proton pumping 4H (matrix) 4H+ (cytosol)
Net equation: NADH + Q + 5H+ (matrix) NAD + QH2 + 4H+ (cytosol)
# of cofactors participate in Complex I:
FMN accepts 2e- (Hydride) simultaneously from NADH & transfers them 1 at a time to 1e- carriers Fe-S clusters.
These electrons reduce membrane-embedded Q(oxidized) to QH2(reduced) in two 1electron steps/CoQ is freely
diffusible in the lipid bilayer & can shuttle 2 reducing equivalents (electrons). The electrons in quinols will be
shuttled to Complex III & eventually to O2 via complex 4.
NADH = carries 2 electrons (Nicotinamide Adenine Dinucleotide made from vitamin Niacin)
FMN = accepts 2 electrons (but can transfer 1 at a time) (Flavin Mononucleotide)
Clusters: can only accept 1e- at a time/ (ferric) Fe 3+ Fe 2+ (ferrous)
Fe = cluster of single iron bound to 4 Cys residues
2Fe-2S = 2 clusters w/ ions bridged by sulfide ions
4Fe-4S = 4 clusters bridged by 4 sulfide ions
Protons pumped per NADH: the electron flow results in pumping 4 protons to inter-membrane space and uptake
of 2 protons from mito matrix to QH2
Inhibitors
Amytal (barbiturate drug/sleeping pill): Non-selective CNS depressants that are primarily used as sedative
hypnotics. Binds to Quinone site of Complex Istops H+ pump ATP synthesis energygo to sleep
Rotenone (insecticide from plant): isolated from plant roots & people catch fish by releasing root extracts to H2O
Myxothiazol (antibtiotic)/Piericidin A (antibiotic)
All inhibit e- transfer rxns from Fe-S clusters to Qblocking the overall process of oxidative phoshorylation
Diseases
Leber Hereditary Optic Neuro/Mito Encephalomyopathy w/ Lactic Acid&Stroke-like Episodes/Leigh Syndrome

Complex II (aka Succinate Dehydrogenase/Succinate CoQ Reductase=SQR): SMALLEST
4 subunits
2 Reactions:
Rxn1: Succinate + FAD(enzyme-bound)fumarate + FADH2(enzyme-bound)
Rxn2: FADH2 + QFAD + QH2
Net Rxn: Succinate + Q fumarate + QH2
FAD (covalently bound) and non-heme 2Fe-2S & 4Fe-4S are the cofactors for Succinate Dehydrogenase.
FADH2 synthesized from the oxidation of succinate & still bound to enzyme (SDH), undergoes further
oxidation/reduction rxns to pass e- to Q
NO proton pumping: so the favorable energy of rxn is not conserved for ATP synthesis. But the energy passed
down to Q will be used by Complexes III & IV to pump protons out of the matrix, contributing to ATP
synthesis
Inhibitor: Malonate
Diseases: Leigh Syndrome & certain tumors result from defects in Complex II due to mutations coding the protein

Complex III (aka Cytochrome b-c1 Reductase/CoQ-cytochorme c Oxidoreductase)
Homodimer of polypeptides: identical Monomers of 11 subunits
Each monomer has 2 cyt b hemes, Heme bL & bH, and a single heme c1. In the center have 2Fe-2S cluster=
Rieske Fe-S Center: 1 Fe is coordinated by 2 Histidine Ligandsparticipate in e- transfer rxns
2 binding sites for Q: P(Qo)=cytoplasmic site & N(Qi) matrix site=. Sites participate in oxidation of CoQH2,
by Qcycle explains the mechanism of QH2 oxidation & Cyt c reductionQcycle results in loss of 2
protons from matrix
Complex III couples transfer of e- from QH2 (ubiquinol) to Cyt c w/ the transport of protons from matrix to
intermembrane space
QH2 + 2Cyt c(oxidized) + 2H+ (matrix) Q + 2Cyt c(reduced) + 4H+ (cytosol)
The energy of oxidation of QH2 is converted into EP by pumping 2 protons from matrix to intermembrane
space=less than half of what Complex I does so ATP amount made from this energy is half too
Cyt c(oxidized) = Fe3+ & Cyt c(reduced) = Fe2+
Protons pumped per NADH: each half of the Q cycle pumps 2 protons out of the matrix = total of 4H+
Inhibitors:
Stigmatellin & Myxothiazol bind to Psite, blocking ETC rxns btw QH2 & Cyt c. (PMS)
Antimycin A: produced by Streptomyces bacteria/used in fish poison (piscicide) & Ilicicolin bind to Nsite blocking
the enzyme too. (NIA)
Disease: Encephalomyopathymuscle & nervous system dysfunction

Complex IV (aka Cytochrome c Oxidase=COX)
13 subunits (integral membrane protein w/ distinct polypeptides)
Prosthetic groups: CuA/CuA center, CuB center, Heme a, Heme a3 (all are 1e- acceptors & participate in
electron transfer rxn). Heme a-CuB site=site of reduction of molecular O2 to H2O (where O2 binds).
Cyt c is 1e- carrier
e- carried by Cyt c (from reduction of QH2 by complex III) are shuttled to Cyt c Oxidase (Complex IV). The
e- are then used to pump protons out of matrixconsumed by O2 resulting in H2O (reduction of O2)
2 Reactions
Rxn1: Reduction of O2 to H2O
4 Cyt c(reduced) + O2 + 4H+ (matrix) 4 Cyt c(oxidized) + 2H2O
Rxn2: Pumping of 4 protons out of Matrix
4 H+ (matrix) 4 H+ (cytosol)
Net Rxn: 4 Cyt c(reduced) + O2 + 8H+ (matrix)4 Cyt c(oxidized) + 2H2O + 4H+ (cytosol)
Overall rxn results in loss of 8 protons from matrix creating a pH gradient. 4 protons are stoichiometric and the
other 4 result from cytochrome oxidase acting a proton pump
Cytochrome c Oxidase Rxn Cycle
Rxn mechanism of 4 e- reduction of O2 to H2O in cytochrome oxidase
Cycle begins & ends w/ all prosthetic groups in their oxidized forms
Cytochrome C is in the Inter-membrane space, so the active site for this rxn w/ Cytochrome C is on the
cytosolic side of the inner membrane
4 cyt c molecules donate 4e-, which, in allowing binding & cleavage of an O2 molecule, also makes possible
the import of 4 protons from matrix to form 2 molecules of H2Owhich are released from the enzyme to
generate the initial state.
1) 2 molecules of cyt c sequentially transfer e- to CuA/CuA to Heme a Heme a3 then to CuB (both reduced)
(Heme a3 has a high affinity for molecular O2 & binds it)
2) Reduced CuB & Fe in Heme a3 bind O2 (reducing it), which forms a Peroxide Bridge
3) 2 more Cyt c molecules add 2 more e- reducing O2 resulting in cleavage of Peroxide Bridge & uptake of 2
protons
4) The addition of 2 more protons leads to H2O release (from rxn btw Hs & OHs bound to Heme a3 & CuB)
Protons pumped per NADH: 2 total because only use O2.
Inhibitors: Cyanide (CN-), Azide (N3-) & Carbon Monoxide (CO) inhibit Cyt c oxidase by binding to Heme
a3/CuBthey block O2 from binding to these prosthetic groups! (CoCA)
Diseases: Encephalomyopathy & Myopathy

NADH oxidation via Complex I, III and IV:
Complex I: NADH + Q + 5H
+
(m) NAD
+
+ QH
2
+ 4H
+
(c)
Complex III: QH
2
+ 2 Cyt c (ox) + 2H
+
(m) Q + 2Cyt c (red) + 4H
+
(c)
Complex IV: 2 Cyt c (red) + 4H
+
(m) + O
2
2 Cyt c (ox) + H
2
O + 2H
+
(c)
---------------------------------------------------------------------------------------------------------
NADH + 11 H
+
(m) + O
2
NAD
+
+ H
2
O + 10 H 10 H
+ +
(c) (c)
FADH
2
oxidation via Complex II, III and IV:
Complex II: FADH
2
+ Q FAD + QH
2
Complex III: QH
2
+ 2 Cyt c (ox) + 2H
+
(m) Q + 2Cyt c (red) + 4H
+
(c)
Complex IV: 2 Cyt c (red) + 4H
+
(m) + O
2
2 Cyt c (ox) + H
2
O + 2H
+
(c)
---------------------------------------------------------------------------------------------------------
FADH
2
+ 6 H
+
(m) + O
2
FAD + H
2
O + 6 H
+
(c)
Summary of ETC:
where m= matrix, c= cytosol


Know how the energy from the oxidation-reduction reactions in the electron transport chain is saved for the
synthesis of ATP (formation of a proton gradient across the membrane).
Some of the energy released from the oxidation of NADH & reduction of O2 is conserved as a form of
electrochemical gradient by proton pumping actions of Complexes I, III, & IVthe energy conserved @ these 3
steps is utilized by mitochondrial ATP synthase to make ATPs

Understand the Thermodynamics of the ETC.
Know how the standard free-energy change (G) and change in standard reduction potential (E) are related in
oxidation reduction reactions.
Half-rxns Standard Reduction Potential
O2 + 2H + 2e H2O E = +0.82 V
NAD + H + 2e NADH E = -0.32 V
Full rxn is (a)-(b) = (c)
NADH + H + O2 H2O + NAD
E = +1.14V


If rxn is exothermic (energy released)= +E.
G = nF E (n = # of electrons & F = Faradays constant = 96, 485 C/mol) 4.185 J/cal 1000 = kcal/mol
The E of rxn for NADH oxidation & O2 reduction is 1.14volts or G=-52.6 kcal/mol (this energy drives the e-
transport & proton pumping)
NADH has a negative reduction potential (strong reducing agent wants to donate e-)
O2 has a positive reduction potential (strong oxidizing agent wants to accept e-)

The flow of Electrons proceed down a thermodynamically favorable pathway
(NADFMNCoQCytbCytc1CytcCytaCyta3O2)







Lecture 17
ATP Synthase Structure:
Fo: integral membrane component a/b/c subunits
Proton turbine
O= oligomycin-sensitive
aproton channel/c subunits form a concentric c-ring in membrane
F1: peripheral membrane protein ////
ATPase (enzyme activity)
subunit inserted into 33 hexamer ring (catalytic unit) which is fixed to the a subunit via subunit b (stator)
breaks symmetry in the 33 hexamer ring
Stationary part (stator): , b2, , & 33
Moving part (rotor):c-ring & stalk (tightly attached to c-ring) rotation of this is propelled by the proton
gradient
proton enters from the intermembrane space into the cytoplasmic half-channel to neutralize the charge on Asp61 in
csubunit (the proton is attracted to the negative charge of the carboxylate)with the charge neutralized the c ring
can rotate clockwise by 1 c subunit moving the Asp residue out of membrane into matrix half-channel this proton
can move into the matrix, resetting the system to its initial state. 10protons/revolution (10 subunits).

Understand the binding-change mechanism of ATP synthesis
Rotation of the stalk interconverts the 3 subunits btw 3 diff conformations. Rotates CW from top & CWW from
bottom
360 rotation of the subunit will lead to synthesis and release of ATP from each subunit
1) Open form: ATP release/ADP + P bind loosely
2) Loose form: ADP + Pi are bound tightly. ATP in T form can not be released until site is filled by ADP &
Pi
3) Tight form: ATP present in equilibrium w/ ADP + Pi. Exchange occurs btw H2O & Pi. Energy (from
proton gradient) is required to change its conformation to O form so ATP can be released.
OLTOLT

Know Common Inhibitors of ATP Synthase
1) F1 Inhibitor Protein: naturally occurring protein in the mitofunction is to prevent ATP hydrolysis by
ATP Synthase under conditions that the mito is ATP rich & not synthesizing ATP

Chemical Inhibitors: both natural and syntheticinhibit both synthesis & hydrolysis of ATP
2) Aurovertin Bsite of action F1a poisonous mushroom binds to subunit inhibiting ATP synthase
3) Dicyclohexyl-carbodiimide (DCCD) site of action Foreacts to free caboxylate groups that are in
hydrophobic environments forming a covalent bond
4) Oligomycinsite of action Fobinds to subunit.
Both (D&O) prevent the influx of protons by ATP synthase

Carrier systems of the inner mitochondrial membrane
5 Types
1. ATP/ADP Translocase:
Function: shuttle ATP of out (export) & bring ADP in (import) to mitowhen ADP is brought in it
is phosphorylated then exported out
Antiport system
Electrogenic (-1) due to ATP-4 & ADP-3
**2 inhibitors: 1) Atractyloside (plant glycoside): binds to translocase when it is open to
cytoplasmic side; 2) Bongkrekic (antibiotic from mold): binds when it is open to matrix side
Combo of Translocase/Pi carrier/ATP Synthase exist as complex called ATP synthasome.
2. Phosphate Carrier:
Function: import H2PO4 w/ symport of a proton H+
Symport
Electroneutral but alters the proton gradient by bringing a proton into the matrix
Combining the ADP & Phosphate carrier together, Phosphate & ADP (substrates for ATP synthesis)
are brought into the mito, which effectively brings along a single H+
3. Other Carriers
Include those of Pyruvate (Pyruvate/OH-), Dicarboxcyclic Acid (Phosphate/Malate),
Tricarboxycyclic Acid (Malate/Citrate+H) & Amino Acid Carriers
4. Malate-Aspartate Shuttle (liver & heart)
Function: shuttles reducing equivalents from NADH, via OXA reduction to Malatethings to
know listed below:
Aspartate is involved in transamination rxn w/ -KG to form OXA & Glu both in matrix & cytosol
OXA is reduced by NADH to form Malate
Malate transverses the membrane
NAD+ is reduced to NADH w/ oxidation of Malate back to OXA
NADH is effectively, but not actually, transferred across the membranethe reducing equivalents
are transferred across the membrane
5. Glycerol Phosphate Shuttle (present in brain)
Function: shuttle reducing equivalents for FADH2
Glycerol-Phosphate is formed from the reduction of Dihydroxyacetone Phosphate by NADHwhich
is able to transverse the mito membrane
Once inside the mito it is converted to DHAP via FAD+ linked enzyme (G3PDH) forming FADH2
Net loss of 1 ATP per NADH because FADH2 is @ a lower potential than FMN from NADH
dehydrogenase & only provides enough enrgy for 2 ATPs via ETC
So the NADH made in the cytoplasm during glycolysis go into the matrix by shuttles (not by carriers)

Know how many ATP molecules can be synthesized from the complete oxidation of a glucose molecule
30 (glycerol phosphate) 32 (malate-aspartate)
Note: Anaerobic metabolism yields only 2 molecules of ATPone of the effects of endurance exercise is to increase
the num of mito & blood vessels in muscle thus increasing the extent of ATP generation by Ox/Phos

P/O Ratio:
Number of Pi consumed per oxygen atom in ATP synthesis by mito
aPi + aADP + O2 + H + NADH (or FADH2) aATP + NAD (or FAD + H) + H2O
a = P/O ratio for NADH = ~2.5 (10/4) & for FADH = ~1.5 (6/4)
Summary of yield of ATP by OxPhos of Various Reducing Equivalents
Glycerol 3-Phosphate (NADHFADH2) 1.5 ATP
Malate-Aspartate (NADH) 2.5 ATP
FADH2 in matrix 1.5
NADH in matrix 2.5

Control of ETC: Respiratory Control
Electrons do not usually flow thru the ETC to O2 unless ADP is phosphorylated @ the same
timeRespiratory Control.
In active muscle: ADP = OxPhos rateADP determines the OxPhos rate
State 4: typical resting state before adding ADP or after ADP is all used up
State 3: Actively respiring (active movement and use of OxPhos)
RCR: Respiratory Control Ratio=the ratio of O2 consumption rate in state 3 & 4. Actively respiring mitos
will have a high RCR.
ADP Level also affects the rate of TCA:
o In Resting muscle ADP is low so NADH & FADH2 oxidation is slow under this condition,
NAD+ & FAD+ level is low so TCA slows down.
o In Active muscle ADP is high so NADH & FADH2 will be used up more to make ATP
under this condition, NAD+ & FAD+ level will be higher so TCA speeds up
Conclusion: ETC works only when ATP needs to be synthesized!
Understand the actions of uncoupling proteins and chemical uncouplers
Heat generation by mito: Nonshivering Thermogensis by uncoupling Proteins.
In animals, brown fat is a specialized tissue that generates heatuncoupling proteins dissipate the proton
gradient, generating heat w/o ATP synthesis.
UCP1 (aka thermogenin): present in the inner mito membrane forms a pathway for the flow of protons from the
Cytoplasm to Matrix energy of proton gradient is released as heat. This dissipative proton pathway is activated
when the core body temp begins to fall, the release of hormones triggered by the temp drop leads to liberation of free
fatty acids from triacylglycerols that in turn activate UCP1.
2,4-dinitrophenol (DNP): uncouples the tight coupling of electron transport & phosphorylation in mito. It is able to
cross the membrane as a charged species & has a proton w/ a pka around 7.0
ETC keeps running w/ uncoupler because it is trying to make up for ATP deficit the loss of respiratory
control leads to increased O2 consumption and oxidation of NADH.

Reactive oxygen species (ROS) generation and their roles are in tissue aging and apoptosis.
In hypoxic cells (stroke/heart attack) there is an imbalance btw the input of electrons from fuel oxidation in mito
matrix & transfer of electrons to molecular O2 this leads to formation of Reactive Oxygen Species.
Complex I & III are the primary sites of ROS generation: in Complex I, superoxide is produced in the bound
flavin facing the matrix side. In Complex III, superoxide is formed @ the ubiquinol oxidation site (Qo site, center P)
facing the intermembrane space.
Superoxide: is produced when there is a transfer of 1 electron to molecular O2
Peroxide: produced when there is a transefer of 2electrons to O2
Hydroxyl Radical (Fenton rxn): Fe2 + H2O2 Fe3 + OH + OH
produced when peroxide reacts w/ reduced iron. This radical is very reactive can damage DNAexample: guanine
base reacts w/ 2 hydroxyl radicals to forms H2O & 8-oxoguanine which can incorrectly base pair w/ adenine
during DNA replication resulting in a G-C to T-A base pair substitution=mutation in amino acid in protein

Oxidative damage by ROS has been implicated in apoptosis (programmed cell death)/ cellular injury during
ischemia & reperfusion/ aging process / pathophysiology of neurodegenerative diseases including Parkinson,
Huntington & Alzheimers/ cellular signaling

Defense Mechanisms against the ROS
1) Superoxide Dimutase (SOD): catalyzes the degradation of superoxide radicals into hydrogen peroxide
(H2O2) & O2. 2 forms: manganese-containing in mito (MnSOD) & copper-zinc dependent in cytoplasm
(Cu/ZnSOD).
Amyotropic Lateral Sclerosis (ALS) or Lou Gehrigs Disease: is caused by a mutation in gene coding for
cytosolic SOD it is a rapidly progressive, invariably fatal neurological disease that attacks nerve cells
responsible for controlling voluntary muscles.
2) Catalase: a ubiquitous heme protein that breaks the H2O2 into H2O & O2
3) Glutathione Peroxidase: also scavenges H2O2
4) Antioxidant Vitamins E & C: exercise ROS defense mechanisms

Role and mechanism of apoptosis in maintaining tissue homeostasis.
During development or for the maintenance of health of the body, cells are born & die constantly
Cell death occurs according to well-orchestrated pre-programmed processes (Programmed Cell Death or
Apoptosis)
Too much cell death can lead to: Neurodegeneration/Immunodeficiency/Infertility
Too little cell death can lead to: Cancer/Autoimmunity
Apoptosis: may be triggered by an external signal, acting @ the plasma membrane receptor (extrinsic pathway), or
by internal events such as DNA damage, viral infection, oxidative stress from accumulation of ROS, or heat shock
(intrinsic, or mitochondrial pathway)
Mitochondrial apoptosis has 3 phases:
1) Pre-mito Initiation phase: cells recognize danger signals & activate death-inducing pathways while
attempting to cope w/ stress by activating pro-survival mechanism
2) Integration Phase: Pro & anti-apoptotic metabolic cascades converge on mito & if legal signals predominate,
mito membranes are permeabilized (MMP) when MMP is permanent & affects mito significantly, cells are
irreversibly committed to death
3) Post-mito Execution Phase: MMP leads to mito transmembrane potential dissapiation (no proton
concentration gradient across the inner-membrane), respiratory chain uncoupling, ROS overproduction, ATP
synthesis arrest & release of protein in the intermembrane space into cytoplasmcell death
MOMP (Mitochondrial Outer Membrane Permeabilization): Cell death signals: ROS/Ca2+ /Lack of survival
signals/death ligands/various stresses. Bcl-2 family forms a pore on outermembrane onlypermeabilizing itit
spills out Cytochrome C & other factors that bind to other proteins forming proteasesactivating caspasescell
death.
PTPC (Permeability Transition Pore Complex): Opening in the inner-membraneoccurs under hypoxic
conditions (ischemia due to heart attack/stroke) followed by reperfusionwhen blood is supplied again it triggers
PTPC H2O & small solutes rush into mitorupture occurs due to osmosiscell death/necrosis

Ischemia: Inadequate blood supply (circulation) to a local area due to blockage of the blood vessels to the area.
Hypoxic: deficiency in the amount of O2 reaching body tissues






































Lecture 18
Understand what the mitochondrial genomes are made of.
For the function of mito ~1500 genes are predicted to code for various factors:
Among them, 37 genes are encoded by mito DNA (mtDNA)exists as circular double-stranded DNA.
2-rRNAs
22-tRNs
13-polypeptides for OxPhos
Gene for complex II not present in mito (7 for I=ND1,2,3,4,4L,5,6)(1 for III=Cytochrome b)(3 for
IV=COI,II,III)(2 for ATP Synthase=ATPase6 & 8)
Remaining 99% of genes are encoded by nuclear DNA
Each mito has 2-10 copies of its own DNA, separate from the nuclear DNA

Types of Mutations:
1) DNA insertion or deletion mutations: frameshift mutations/chain terminationtotal or partial loss of genes
2) DNA base substation mutations
A) In protein-coding genes: amino-acid substitution, or polypeptide chain termination
B) In tRNAs or rRNAs: protein synthesis compromised globally
Genetics of Mito Dysfunction
By mtDNAsmaternal pattern inheritance in phenotypes (w/ large variability)
By nuclear DNA mutationsMendelian pattern
If both are mutated then it will be hard to predict which way it will work

Know how mitochondrial DNAs are replicated and how they are inherited.
Mito are inherited maternally: Oocyte has 100,000/sperm has 50-75
mtDNA are prone to mutations can be exposed to ROS produced by the respiratory chaindefects in mito DNA
accumulate over time during the lifetime of each individualone theory of aging is that the gradual accumulation of
defects w/ increasing age is the primary cause of many symptoms of aging.

Female germ-line filter is:
maternally inherited mito DNA has a high mutation rate & mito DNA base substitution or deletions have been
reported in a variety of inherited degenerative diseases including Myopathy, Cardiomyopathy & Neurological &
Endocrine disorders
-Studies suggest that oocytes containing mito w/ severe mutations in their mito DNA are selectively eliminated by
apoptosis during oogenis, however the filter is not perfect it will select healthy oocytes too.

Heteroplasmy
When an oocyte containing certain % of mutant mito is fertilized & undergoes many cell divisions during embryonic
development, the resulting somatic cells can harbor mutant mito in varying proportionsmito are segregated to the
daughter cells randomly.
Individuals w/ the same affected mito may have disease symptoms of diff severity in diff organsit is repeatedly
demonstrated that mtDNA mutations can produce markedly diff symptoms among members of affected family

Simple genetic pattern of the mitochondrial diseases: affected mito genome is transmitted from mother to offspring
whn then shows the disease









Representative syndromes/diseases caused by the pathogenic mitochondrial DNAs.
Disease Mutation Symptoms
DEAF: deafness rRNA
NARP:
Neurogenic muscle weakness
Ataxia (failure of muscular coordination)
Retinis Pigmentosum (slow retinal deterioration)
ATPase low 10-20%
(T8,9993Gleu to Arg change in
subunit 6marked instability in
ATPsynthase, ATP synthesis,
ROS production)
muscle strength & coordination
regional brain degeneration
retinal degeneration
seizures
dementia
sensory neuropathy
developmental delays
Leigh Syndrome
Lethal childhood disease
ATPase high 70%

details same as NARP
Caused by nuclear or mtDNA
mutations
Degenerative neurological
condition lesions in basal ganglia,
thalamus & brainstemresulting
in developmental delay
seizures
uncontrolled eye movements
breathing abnormalities
LHON
Leber Hereditary Optic Neuropathy
Complex I genes
ND4 G11778A (69% of cases)
His to Arg replacement @ residue
340 in subunit 4 in Complex I
Mid-life (27yrs), sudden onset
blindnessmaternally inherited.
Caused by death of optic nerves
More common in males than
females
May also have cardiac problems
& behavior abnormalities
MERRF
Myoclonic Epilepsy & Ragged Red Fiber
tRNA & rRNA
tRNA Lys A8344G mutation results
in defective tRNA Lysoverall
decrease in mito protein
synthesisOxPhos compromised
(Complex I & IV have the greatest
num of mito encoded subunits so are
the most affected)
Uncontrollable muscular jerking
(myoclonic epilepsy)
Muscles less effective
Mito Myopathy (RRF) over-
accumulation of mito makes them
look ragged
Type II Diabetes Low level 10-30%
tRNA Leu A3243G
Non-insulin dependent diabetes
MELAS
Mito Encephalomyopathy
Lactic Acidosis
Stroke-like Episodes
High level 70%
tRNA Leu
(generally associated w/ complex I
defects)
Worsen w/ age (degenerative)
Short stature
Cardiomyopathy
Mito encephalomyopathy
Lactic acidosis

Threshold hypothesis
Hypothesis for how age-related progression of OxPhos diseases occur
w/age the amount of defective mito DNA increases for both normal & patients
But the patient starts off w/ a larger % of damaged DNA due to disease
The relative amount of damaged DNA is proportional to tissues ability to perform OxPhos (some tissues like
optic track require higher capacity than others soThreshold is diff for diff organs
Both normal & patient have progressive loss in OxPhos accompanied by a fixed progression of defects in
tissues w/ age.
Difference is the patient realizes the defects @ an earlier age than normal
w/ added mutations the capacity for the cell to make ATP decreases w/ no ill effect until a threshold level is
reach at this time the tissue or organ displays a phenotype
Ex. Symptoms in patient show up @ age 45 in comparison to normal person @ age 90




Lecture 19
Monooxygenases (Mixed Function Oxidases/Oxgenases): incorporate 1 oxygen atom into substrate & one to H2O
R-H (substrate) + O2 + ZH2 (co-substrate) R-OH + H2O + Z
Co-substrates: NADH / NADPH / FMNH2 / FADH2
Reactions involved:
1) Drug metabolism by Cytochrome P450
2) Synthesis of Tyrosine/Serotonin/Catecholamines-DOPAepinephrine & norepinephrine
3) Cholesterol
4) Vitamin D
5) Nitric Oxide

A1) Cytochrome P450
Monooxygenase ~50kd
Contains Heme that absorbs light maximally @ 450nm (when reduced & CO-bound)
Family of enzymes (57 genes in human)
Present in mito & ER of liver and other tissues
Catalyzes hydroxylation, epoxidation & other modification of hydrophobic (aromatic) compounds (drugs)
for their detoxification & excretion
Catalyzes synthesis of steroid hormones & bile salts
Causes drug interactions

Mechanism of Action:
In order to hydroxylate the substrate P450 activates oxy molecule using its iron containing heme.
For it to function it also requires NADPH which transfers 2 high potential electrons to flavoproteinwhich
tranfers them 1 @ a time to adrenodoxin (a non-heme iron protein) or to CYP450 reductase which then transfers 1
electron to reduce the Ferric (Fe3+) to Ferrous (Fe2+)
1) substrate binding (RH)
2) e- transfer for reduction of iron: ferric to ferrous as stated above (Fe3+ Fe2+)
3) binding of oxygen molecule to heme (w/o the addition of e- P450 will not bind oxygen)
4) adrenodoxin adds in 1 more electron that break O=O bond to O-O bond
5) add in 2 H+, one oxy protonated then release H2O
6) Hydroxylation & release of product

Also responsible for:
Hydroxylation of foreign compounds, which increases their solubilityfacilitating their excretion.
It metabolizes ibuprofen & caffeine convert to soluble formrapid excretionlowering efficacy
Steroid hormones (testosterone/estrogens) are derived from cholesterol by P450 action
Acetaminophen toxicity by P450: liver toxicity is observed w/ large doses of acteminophen (pain killer)
P450 isozyme oxidizes it to N-acetyl-p-benzoquinone imineresulting compound is a conjugated
glutathione. With larger doses of drug, the liver con of glutathione drops dramaticallyliver is no longer
able to protect itself from this reactive compound
Metabolism of benzo[a]pyrene by P450: it is found in coal tar/car engine exhaust fumes/smoke from any
burning organic material (cigs & charboiled food/toast). In the body P450 metabolizes it to
(+)benzo[a]pyrene-7,8 dihydrodiol-9,10 epoxide (most carcinogenic compound aroundreacts covalently w/
DNA)

A2) Tyrosine Synthesis
Phenylalanine hydrolase catalyzes rxn.
The oxidant is O2 & reductants are Phe & NADH
Cofactor: Tetrahydrobiopterin acts as the initial reductant in rxn & is oxidized to quinoid dihydrobiopterin
(which can be converted back to THB by its reduction w/ NADH)
Phenylketonuria (PKU)
Most common disease due to loss of phenylalanine hydroxylase. 97% due to recessive mutation in gene encoding
phenylalanine hydroxylase & 3% due to recessive mutation in genes whose products are required for synthesis or
reduction of biopterin.
High concentration of phenylalanine in blood leads mental retardation (1%)/therarpy=low phenylalanine diet(casein
from milk). Phenylpyruvate (aka phenylketone) is produced by deamidation of phenylalanine & removed by urine
when phenylalanine builds up.

A3 Serotonin Synthesis
Tryptophan is hydroxylated to 5-hydroxytryptophana precursor for neurotransmitter serotonin. Low level of
serotonin or compromise d signaling by compound can influence mood, leading to depression. Tryptophan hydrolase
uses tetrahydrobiopterin same as phenylalanine hydrolase.

A4) Catecholamine Synthesis
(hormones that mediate stress response). Tyrosine hydrolase converts Tyrosine to DOPAprecursor for
norepinephrine/epinephrine. Parkinsons disease is caused by insufficient formation & action of dopamine in the
brain.

A5) Cholesterol & Vitamin D
Squalene monooxygenase catalyzes the hydroxylation of squalene to cholesterol using NADPH & O2

In Vit D3 (calcitriol) synthesis, 2 hydroxylation reactions occur. 1) in the liver microsome & 2) In kidney
mitochondria by monooxygenases

A6) Nitric Oxide Synthesis
Nitric oxide (NO is a free radical gas @ room temp) is an important messenger in signaling pathways in vertebrate
animal cells. For example, NO stimulates mito biogensisthe free radical gas is produced from arginine in a
complex rxn catalyzed by NO Synthase. NADH & O2 are required for the synthesis of NO. 1
st
rxn arginine is
hydroxylated @ guanidinium group/2
nd
rxn: a 2
nd
hydroxyl group is addedwhich reacts to release NO & citrulline.

Dioxygenases: catalyze 2 hydroxylation rxns utilizing O2 & reducing agent. Incorporate 2 oxygen atoms into
substrate

R (substrate) + O2 R-O2
Co-substrates: various reducing agents such as ascorbic acid
Reactions involved:
1) Prostaglandin synthesis (COX-1, 2): Target of NSAIDs
2) Degradation of Phenylalanine, Tyrosine
3) Synthesis of Collagen, Retinal, Vitamin A1

B1) Cyclooxygenases in inflammation & NSAIDs
Dioxygenases are important for the synthesis of prostaglandins. Cyclooxygenase (COX), aka Prostaglandin H2
Synthase converts Arachindonate to Prostaglandins, beginning w/ the formation of Prostaglandin H2 (PGH2)the
intermediate precursor of many other prostaglandins & thromboxanes.
Mammals have 2 isoenzymes/isoforms of prostaglandin H2 synthase (COX-1 & COX-2)which are similar in
sequence & structure. COX-1: constitutively expressed & involved in prostaglandin biosynthesis in response to
hormone stimulation COX-2: an inducible enzyme that is expressed transiently in response to growth factors, tumor
promoters, or cytokinescatalyzes the production of prostaglandins that mediate inflammation, pain & fever.

NSAIDS (non-steroidal anti-inflammatory drugs): aspirin or ibuprofen inhibit COX-2 which is induced w/ injury
or from trauma that results in an inflammation stimulus. Aspirin inactivates COX-2 by acetylating the active site of
the enzyme inactivating it so this reduces fever & swelling. COX-2 inhibitors inhibit COX-1 which is responsible
for housekeeping prostaglandins, like E2, which is used for kidney function, & I2 which is used for stomach
protection.
Specific drugs made for COX-2 did not effect kidney/stomach but did result in increased heart attack/stroke
(Celebrex{Celecoxib} & Vioxx

B2) Dioxygenases in Phenyl Alanine/Tyrosine catabolism
In synthesis & degradation of Tyrosine1 monooxygenase & 2 dioxygenases are required. 1
st
for formation of
homogentisate from p-hydroxyphenylpyruvate & 2
nd
for the formation of 4-maleylacetoacetate from homogentisate.
In Last rxnthe result is a cleavage of aromatic ringnearly all cleavages of aromatic rings involve dioxygenases.

Alcaptonuria is due to a defect in Homogentisate Oxidase resulting in excretion of homogentisate in urine this
autooxidizes the quininewhich polymerizes forming a deep black color in urine of patients w/ the disease (no other
symptoms).

B3) Prolyl Hydroxylase in collage Synthesis & Scurvy
In synthesis of Collagen (structural support that helps bind cells together), both hydroxyl proline & lysine are
required. Hydroxylation rxns occur by dioxygenases that utilize ascorbate (Vit C) as the reducing agent rxn results
in hydroxylation of proline & decarboxylation of -KG forming succinate.
Scurvy results from a deficiency in Vit C people w/ this have internal hemorrhages (which result in black &
white marks on the skin)/bleeding gums/weakness & joint pain.
In the absence of hydroxylproline, the blood capillaries break down & hemorrhaging occurs throughout the body

B4) Dioxygenase in Retinal Synthesis
Dioxygenase is needed for formation of Retinal & Vitamin A from -carotene. In Vit A1 synthesis, -carotene is
hydroxylated & reduced to form vit A1 the rxn breaks C=C bond resulting in 2 molecules of trans-retinal
Retinal is very important for vision while Vit A has many other roles in the cell.

B5) Other Oxidation Rxns in Cell (Ethanol)
Ethanol is oxidized to acetylaldehyde by liver dehydrogenase. Microsome P450 will also oxidize alcohols so upon
large amounts of alcohol, ethanol acts as a competitive inhibitor of P450 & can inhibit their interactions w/ other
drugs, such as barbituates because of this lives of other drugs are increased resulting in an increase in
concentration of these drugs in the bodymay be the reason why alcohol should not be consumed when other drugs
are being taken.
1
st
oxidation product of ethanol is acetylaldehyde (a toxic compound that can cause a severe rxn in the body).
Aldehyde Dehydrogenase rapidly oxidizes acetylaldehyde so that it doesnt build-up.
Antabuse is a drug used for recovering alcoholicsinhibits aldehyde dehydrogenase so that acetylaldehyde can
build up for the physiological response to prevent alcoholics from going back to drinking (risk of too muchdeath)

Methanol is oxidized by the same enzymes as ethanol. The final result is formic acid (toxic & results in decreasing
the blood pH which can lead to blindness). Antidote to methanol ingestion is large amount of ethanolwhich
competes for alcohol dehydrogenase & prevents the oxidation of methanolso excreted from the body

Ethylene Glycol results in the formation of oxalate which is a strong chelator of Ca2+ ions & results in a
precipitate the precipitate collects in the renal tubes & results in kidney failuredeath. Ethylene glycol is a major
component of antifreeze used in automobilesits consumption can be countered by ethanol/alcohol consumption.