Biochemistry Ch 21 377-394
Oxidative Phosphorylation and Mitochondrial Function
ATP generation requires NADH, FADH2, an electron acceptor (O2), mitochondrial membrane,
electron transport chain, and ATP synthase. It is regulated by ATP use
Overview of Oxidative Phosphorylation – energy for ATP synthesis provided by electrochemical
gradient across the inner mitochondrial membrane generated by components of ETC that pump
H+ across inner mitochondrial membrane as they sequentially accept/donate electrons
-final acceptor is O2 which is reduced to H2O
1. Electron Transfer from NADH to O2 – in ETC, electrons (e-) are donated by NADH/FADH2
and passed through (e-) carriers in the inner mitochondrial membrane; each component is
reduced
NADH  NADH:CoQ oxidoreductase (complex I)  coenzyme Q (CoQ)  cytochrome b-c1
complex (complex III)  cytochrome C  Cytochrome C oxidase (complex IV)
-CoQ – lipid-soluble quinone free to diffuse through lipids, transports e- from complex I
to complex III
-Cytochrome C – protein in intermembrane space transfers e- from complex III  IV
-cytochrome C oxidase (complex IV) ha binding site for O2, where it is reduced to H2O
2. Electrochemical Potential Gradient – at each complex, e- transfer is coupled to proton
pumping across membrane
a. Transmembrane movement generates electrochemical gradient w/ 2 components:
membrane potential and proton gradient (called proton motive force)
3. Adenosine Triphosphate Synthase – ATP Synthase is an enzyme that generates ATP
containing 2 portions: (F0) = inner membrane stalk portion with 12 C subunits acts as a rotor
attached to the head by epsilon and gamma subunits, and an (F1) = headpiece composed of
three αβ-subunit pairs. Each β-subnuit – has catalytic site for ATP synthesis. Headpiece is
held stationary by a δ-subunit attached to a b-subunit attached to an a-subunit connected
to the c subunits
a. Influx of H+ through proton channel turns rotor
b. C subunit contains glutamyl carboxyl group extending into proton channel which
collects the H+ to turn the rotor
c. Rotation exposes different proton-containing subunit to open chanel
d. Rotation is completed by attraction of glutamyl group and a-subunit
e. 12 C subunits are hypothesized  12 protons to complete 1 turn of rotor to
synthesize 3 ATP
Oxidation – Reduction Components of ETC – some components involved in redox are flavin
mononucleotide (FMN), Fe-S groups, CoQ, Fe in cytochromes b, c1, c, a, and a3
-reduction potential of each complex is at a lower energy level than previous complex, and so
energy is released as electrons move through each complex; energy used for proton pumping
1. NADH:COQ Reductase – also called NADH dehydrogenase: binds NADH, FMN, Fe-S, and CoQ
-FMN accepts 2 e- from NADH and passes them to Fe-S centers which transfer them to
CoQ or cytochrome b-c1
2. Succinate Dehydrogenase – is a part of TCA cycle and a component of complex II of ETC and
can pass e- to CoQ
-also, ETF-CoQ oxidoreductase accepts e- from ETF which acquires them from fatty acid
oxidation and other pathways
-FAD in succinate dehydrogenase is at same redox potential as CoQ, and so no energy is
released upon e- transfer (complex II) and does not have proton-pumping mechanism
3. Coenzyme Q – only component of ETC not protein bound and can diffuse through lipid
membranes
Fully oxidized form (Q) + e-  semiquinone form (Q*) + e-  Reduced form (QH2)
-CoQ is also called ubiquinone
4. Cytochromes – proteins containing a heme atom bound to porphyrin nucleus; cytochromes of
b-c1 complex have higher energy level than those of cytochrome oxidase; energy is released by
e- transfer between complexes III and IV; Fe in cytochromes are in oxidized Fe3+ state, which
are reduced to Fe2+ after accepting an electron
5. Copper (Cu+) and Reduction of O2 – last cytochrome is cytochrome oxidase, which passes efrom cytochrome C to O2; it contains cytochromes a and a3 and O2 binding site
-O2 must accept 4 e- to be reduced to 2 H2O molecules
-Bound Cu facilitates collection of 4 e- and reduction of O2
Pumping of Protons – energy from redox drives proton transport to intermembrane space
-can be demonstrated using Q cycle for b-c1 complex:
-CoQ accepts 2 H+ at matrix side together with 2e-; it then releases H+ into
intermembrane space while donating one e- back to another component of cytochrome
b-c1 complex and one e- to cytochrome C
Energy Yield from ETC – overall free-energy release is -53kcal from NADH O2, and -41kcal
from FADH2  O2
-NADH donates 2 electrons
-(complex I = 4 protons) (complex III = 4 protons) (complex IV = 2 protons)
-4 protons translocated for each ATP synthesized
-2.5 ATP per NADH, and 1.5 ATP per FADH2
Respiratory Chain Inhibition and Sequential Transfer – in the absence of O2, no ATP is
generated from ox-phos because electrons back up in the chain
-Electron flow MUST be sequential from NADH or FAD all the way to O2 to generate ATP
-complex I cannot pump protons to generate gradient because all CoQ already has electrons
that it cannot pass forward
-cyanide is a respiratory chain inhibitor binds to cytochrome oxidase to prevent transfer at all
complexes and stops respiration
-partial inhibition will reduce ATP synthesis
Cyanide’s Effects on Respiration – CN binds Fe3+ in heme of cytochrome aa3 component of
cytochrome oxidase to prevent e- transport to O2. Cell death rapidly follows, and CNS is
primary target for CN toxicity
-cyanide present in air as HCN, in H2O as NaCN, and foods as cyanoglycosides
-cyanoglycosides present in plants like almonts, pits from fruits
-HCN is released from cyanoglycosides by B-glucosidases present in plant or intestinal
bacteria some are inactivated by liver rhodanase, converting it to thiocyanate
Mitochondrial DNA and OxPhos Diseases – mtDNA is double stranded and circular; encodes 13
subunits of oxphos complexes and encodes rRNA, and 22 tRNAs
-mtDNA undergoes maternal inheritance pattern and accumulate somatic mutations with age
-mitochondria replicate with fission, and mutations can accrue at any time
-disease pathology worsens with age due to accumulated mutations in mtDNA from free radicals
1. Kearns-Sayre Syndrome – mtDNA tRNA and OxPhos genes are deleted; <20 y.o
2. Pearson Syndrome – mt tRNA and oxphos genes deleted affects BONE MARROW
3. MERRF – point mutation in tRNA-lysine of mtDNA (RAGGED RED FIBERS)
4. MELAS – point mutations in tRNA-leucine of myDNA (encephalomyopathy, stroke symptoms)
5. Leigh Disease – missense F0 subunit mutation in ATPase, many symptoms
6. LHON – mutation in NADH dehydrogenase causes acute optic atrophy
-mutations have been reported for mit proteins encoded by genomic DNA
-nuclear respiratory factors NRF-1 and NRF-2 promote transcription of respiratory chain
complexes which could have mutations
Coupling of ETC and ATP Synthesis – e- flow cannot occur faster than protons are used for ATP
synthesis
Regulation through Coupling – when ATP is used by energy-requiring reactions, ADP and Pi
build up, which can bind to ATP synthase, which causes more H+ to flow through ATP synthase
pore from intermembrane space to matrix
-as ADP levels rise, proton flux increases, and electrochemical gradient decreases
-results in increased O2 consumption
-increased NADH oxidation in ETC and increased concentration of ADP stimulate fuel
oxidation like TCA cycle to supply NADH and FADH2 to ETC
Uncoupling ATP Synthesis from Electron Transport – when H+ leaks back into matrix without
going through ATP synthase, they dissipate the gradient without making ATP.
-this is called uncoupling oxidative phosphorylation, and occurs with uncouplers
1. Chemical Uncouplers of OxPhos – also called proton ionophores are lipid-soluble
compounds that quickly transport H+ from cytosol to matrix of inner mit membrane
a. Uncouplers pick up protons from intermembrane space and diffuse through
membrane to release them back to matrix side
2. Uncoupling Proteins and Thermogenesis – uncoupling proteins (UCPs) form channels
through inner mit membrane to conduct protons without using ATP synthase
a. UCP1 (thermogenin) – associated with heat of brown fat tissue; major function
of which is nonshivering thermogenesis
i. In response to cold, sympathetic nerve endings release norepinephrine
to activate lipase in brown adipose that releases fatty acids from
triacylglycerols which activate UCP1 along with reduced CoQ
ii. When UCP1 is activated by fatty acids, it transports protons from
cytosolic side of membrane into matrix without ATP generation
iii. UCP1 = brown adipose, UCP2 most cells, UCP3 skeletal muscle
3. Proton Leak and Resting Metabolic Rate – low level of H+ leak across inner mit
membrane occurs in mitochondria all the time, and 20% of resting metabolic rate is
expended to maintain electrochemical gradient dissipated by basal protein leak
Transport through inner and outer mitochondrial membranes – most new ATP needs to be
transported out of mitochondria, and ADP-Pi-pyruvate, metabolites need to go in
Transport through Inner Mitochondrial Membrane – inner mit membrane is a tight
permeability barrier to all polar molecules such as ATP, ADP, Pi, pyruvate, Ca, H+ and K+
-ions transported by protein translocases using active transport through proton gradient or
electrochemical gradient
-ATP-ADP translocase (ANT) – transports ATP from mit matrix to intermembrane space in 1:1
ratio because ATP has 4 negative charges, and ADP has 3, so exchange is promoted by
electrochemical potential
-similar antiports exist for many ions
-inorganic Pi and pyruvate are transported on SYMPORTS with protons
-Ca uniporter is driven by electrochemical potential, which is negative on matrix
and positive on cytosolic side
Transport through Outer Mitochondrial Membrane – outer membrane permeable to
compounds with molecular weight of 6kDa because it contains nonspecific pores called voltage
dependent anion channels (VDACs) formed by mitochondrial portins
-VDACs form a B-barrel with nonspecific water-filled pore in center
-pores are OPEN at LOW transmembrane potential with preference for PO4, Cl,
pyruvate, citrate, and adenine
Mitochondrial Permeability Transition Pore (MPTP) – mitochondrial permeability transition
involves opening of MPTP through inner mit membrane and outer membranes at sites where
they form a junction
-components are ANT, VDAC, and cyclophilin D
-ANT is normally closed pore that functions in 1:1 ATP:ADP exchange, however
increased matrix Ca, excess phosphate, or ROS can open the pore
-ATP on cytosolic side of pore can close pore
-MPTP can be opened by ischemia (hypoxia) which results in lack of O2 for maintaining proton
gradient and ATP synthesis
-without proton gradient, ATP synthase runs backwards to hydrolyze ATP to restore gradient
-lack of ATP for maintaining low Ca can contribute to pore opening, and as anions/cations enter
matrix, mitochondria swells to cause cell death and necrosis
Mitochondria and Apoptosis – loss of mitochondrial integrity initiates apoptosis
-intermembrane space contains procaspases 2,3, 9 which are proteolytic enzymes as zymogens
-it also contains apoptosis-initiating factor (AIF) and caspase-activated DNase (CAD)
-once AIF is inside nucleus, it initiates chromatin condensation and degredation
-cytochrome C can also be released into intermembrane space to initiate apoptosis if in cytosol
-Bax allows entry of ions into intermembrane space and rupture of mitochondrial membrane to
allow proteins like cytochrome C to go through.
-thyroid hormones increase level of UCP2 and UCP3 to increase heat production