8. oxidative phosphorylation

oxidative phosphorylation

use of generated energy to produce ATP

ETC coupled with ATP synthesis

energy production during ETC

Electrons flow from NADH and FADH2 through a series of carriers to reach O2

Energy produced during the transfer of electrons in the electron transport system is used to pump protons into the intermembrane space

Energy produced when these protons reenter the mitochondrial matrix is used to synthesize ATP

Transport of reducing agents

Inner membrane lacks an NADH transporter and cannot enter the mitochondria

G3P + malate shuttle

Glycerol 3-P shuttle

NADH → FADH2

Electrons are transferred from NADH to DHAP by cytosolic glycerol 3-P dehydrogenase

Glycerol 3-P is oxidized by the mitochondrial isoenzyme and FAD is reduced to FADH2

Malate shuttle

NADH → NADH

Oxaloacetate is reduced to malate with use of NADH

Malate enters the mitochondria and is oxidized to oxaloacetate with reformation of NADH

ATP/ADP transport

IMM requires special carriers to transport ADP and phosphate from cytosol into the matrix

Adenine nucleotide antiporter imports 1 ADP from cytosol, while exporting 1 ATP into the cytosol

Phosphate transporter carries phosphate from the cytosol into the matrix

Electron transport chain - structure

located in the inner mitochondrial membrane

4 large multiprotein complexes (I – IV)
2 small carriers: coenzyme Q (CoQ) and cytochrome c

Prosthetic groups
■ FAD and FMN: complexes I & II
■ Heme groups: complexes III & IV
■ Copper ion: complex IV

Carriers transfer electrons between complexes, to finally combine with O2 and H+ → H2O

Complex I

NADH:CoQ oxidoreductase (dehydrogenase) is a giant protein
complex embedded in the IMM

Energy is lost with each passing and is used to pump 4H+ from the matrix into the IMS

Complex II

Succinate dehydrogenase oxidizes succinate to fumarate (TCA cycle), with production of FADH2

No energy is lost in this process

NO protons are pumped at this stage

Parallel entry for electrons into the ETC

Electrons are passed to CoQ one at a time

Coenzyme Q (ubiquinone)

quinone derivative from cholesterol

Only lipid-soluble and non-protein-bound component of ETC

mobile carrier of electrons from complexes I and II to complex III

Carries 2 electrons at a time

Complex III

Cytochrome bc1
electrons from ubiquinone → cytochrome b → cytochrome c1 → cytochrome c

Cytochrome c is a mobile electron carrier that brings electrons to complex IV, one electron at the time!

High drop in energy with electron movement → 4H+ are pumped into
the IMS

Complex IV

Cytochrome a+a3 (cytochrome oxidase)

Conducts electrons through cytochromes a and a3, finally reducing one molecule of oxygen

When 4 electrons are available, 4 protons are used to reduce and split O2 to form 2 molecules of H2O

In the process, 2H+ /H20 from the matrix are pumped into the IMS

Reactive oxygen species

Partially reduced oxygen is very unstable and avid for electrons

O2 can accept 4 electrons

Oxygen is progressively reduced in four steps

CoQ can accidentally interact with O2 → superoxide

oxidative stress

Imbalance between the production of reactive oxygen species (ROS) and removal mechanisms

— Lipid peroxidation
– Proteins oxidization and degradation or aggregation
– DNA damage (base oxidation or double strand breaks

Cellular defenses against oxygen toxicity

Enzymes (glutathione peroxidase, catalase, superoxide dismutase)

Antioxidants (vit, A, C, E)

ETC inhibitors

ETC inhibitors block the flow of electrons to oxygen and inhibit ATP synthesis

Complex I : rotenone, barbiturates

Complex III: antimycin A

Complex IV: cyanide (CN-), carbon monoxide (CO)

chemiosmotic theory

The energy needed to phosphorylate ADP to ATP is produced by a flow of
protons against an electrochemical gradient

The proton gradient is established by H+ pumped from the matrix into the IMS using the energy released by the electron transport through complexes I,
III, and IV

The flow of electrons in the ETC is coupled with the flow of protons across
the membrane and the flow of protons is coupled with ADP phosphorylation

ATP synthase

multisubunit enzyme (complex V)

membrane domain (F0)

embedded in the IMM

Rotor

H+ -channe

Extramembraneous domain (F1)

a sphere that protrudes into the matrix

Head = 3 ab-subunit, each b subunit with catalytic site

ATP synthesis

H+ from the IMS reenters the matrix by passing through the H-channel in the Fo domain, driving the rotation of the c ring

This causes conformational changes the ab-subunits of the F1 , exposing the
catalytic site: ADP+Pi → ATP

One complete c ring rotation produces 3 molecules of ATP

Oxidative phosphorylation summary

NADH and FADH2 are oxidized via the mitochondrial electron transport chain

An electrochemical proton gradient is established across the inner mitochondrial membrane

The proton gradient drives ATP synthesis

Inhibitors of electron transport block ATP synthesis

requirements of oxidative phosphorylation

1. Electron donors: NADH, FADH2
2. Electron acceptor: O2
3. Intact mitochondrial membrane
4. Functional ETC components
5. ATP synthase

ATP synthesis inhibitors

ATP synthesis and ETC are coupled in normally functioning mitochondria

If ATP synthase is inhibited or has inadequate supply of ADP:

ATP synthesis is inhibited

O2 will not be consumed

ETC components accumulate in reduced states

what will happen if ATP synthase is inhibited or has inadequate supply of ADP

ATP synthesis is inhibited

O2 will not be consumed

ETC components accumulate in reduced states

Oligomycin

binds to the Fo domain, closing the H- channels and preventing the reentry of H+ into the matrix → inhibited ATP synthesis and blocked oxidative phosphorylation

Uncoupling proteins

in the IMM form channels that allow H+ to reenter the matrix without synthesis of ATP

ATP production decreases and O2 consumption and ETC rate increase

Energy is released as heat in non-shivering thermogenesis

UPC1/ thermogenin

responsible for heat production in the mitochondria-rich brown adipose tissue

Dinitrophenol

lipophilic H+-carrier that disrupts the proton gradient by carrying protons across the IMM

Oxidative phosphorylation disorders

13 proteins involved in OP are encoded by mtDNA and synthesized in the matrix

Mutation rate of mtDNA is 10x greater than nuclear DNA

genetic defects in OP enzymes

Hereditary defects are very rare and result in lactic acidosis and muscle and nerve pathology (tissues with high ATP requirements)

Leber’s hereditary optic neuropathy

complex I defect
bilateral neuroretinal degeneration with optic nerve damage

Leigh syndrome

Fo defect

optic nerve atrophy, hypotonia, ataxia, respiratory abnormality