Electron Transport and Oxidative Phosphorylation

Oxidative Phosphorylation•It is the process by which electrons are carried from reduced cofactors (NADH+/ QH2) are finalled in stepwise manner to oxygen.

•Electrons flow much like electricity in a circuit with free energy being conserved with the formation of proton gradient.

•In the end the investment of reduced cofactors are utilized in the production of ATP

Oxidative Phosphorylation• Reduced cofactors like NADH+ are produced during

Glycolysis, Citric acid cycle and fatty acid oxidation

• During cellular respiration oxidative Phosphorylation chemical energy of these reduced molecules are utilized to produce ATP

• The ultimate acceptor of e- through a series of O/R reactions is the O2 within the mitochondrion

21.1 Biological Oxidative

Mitochondrial Anatomy

• The anatomy of the mitochondrion reflects its role in oxidative Phosphorylation

• The outer membrane is porous and allows the free diffusion of molecules due to the presence of channel protein called porins

• The inner membrane is impermeable to most substances including ions and encloses a space called the matrix

• The inner membrane is convoluted structure which provides greater surface area for the protein complexes of the oxidative Phosphorylation

Mitochondrial Anatomy• The mitochondrion

consists two membranes separated by a compartment called the intermembrane space

• During oxidative Phosphorylation protons are pumped into this compartment

matrix

inner membrane

outer membrane

inter- membrane

space

mitochondrion

cristae

Transport Shuttles • Inner mitochondrial membrane is impermeable to most

molecules, NADH+ produced by Glycolysis in the cytosol must be imported into via biochemical reaction of the malate – aspartate shuttle

• operates mainly in the liver, kidney and heart

• In the skeletal muscle & brain NADH+ is imported into - by Glycerol – phosphate shuttle

• The process is a formal currency exchange between one region of the cell with the other.

• ATP, ADP and Pi also require transport protein

• Substrate level phosphorylation

• Oxidative Phosphorylation

Redox Potential of the components of Respiratory chain

• In the respiratory chain the e- s are transferred from reducing equivalents to a chain of e- carriers which are arranged sequentially

• The e- s flow from more electropositive components to more electronegative components i.e. to more positive Redox potentials

• Hydrogen and e- s move from NAD+/NADH to O2/ H2O

The Electron Transport Chain

Enzyme and Electron Vector

NADH-coenzyme Q reductase ( I )Succinate-coenzyme Q reductase ( II )Coenzyme Q- cytochrome reductase ( III )Cytochrome c oxidase ( IV )

Components of the Respiratory chain• Three major electron transporting complexes

of inner membrane

• These complexes function in re oxidizing the coenzymes ( NAD+/ ubiquinone, e- transferring flavoprotein) that have been reduced by dehydrogenases in the metabolic reactions within the mitochondrion

• The terminal e- acceptor is O2 & the reaction is coupled to ATP synthesis

Components of Respiratory chain• The respiratory chain consists of number of

Redox carriers proceeding from NAD – linked dehydrogenases through flavoprotein and cytochrome to molecular oxygen

• Certain substrates (fumarate/ Succinate) comparatively of their more positive Redox potential are directly linked to flavoprotein Dehydrogenase further linked to cytochrome to molecular oxygen

Overview of the Electron Transport Chain ( Respiratory Chain )

Components of Respiratory chain• Ubiquinone (Q/ Coenzyme Q) links

flavoprotein to Cytochrome b (member of cytochrome chain with lowest Redox potential)

• Ubiquinone acts as a mobile component of the respiratory chain that collects reducing equivalents from more fixed flavoproteins and passes them to cytochromes

Components of Respiratory chain

• Next component is the Iron – sulfur protein (Fe-S – a non heme protein) associated with flavoprotein and cytochrome b

• Electrons flow through a series of cytochromes in order of increasing Redox Potentials to molecular oxygen

• The terminal cytochrome aa3 (cytochrome oxidase) is responsible for the final combination of reducing equivalents with molecular oxygen.

Components of Respiratory chain

• It has a high affinity for O2 thus allowing the respiratory chain to function at its maximum.

• This is the only irreversible reaction in the chain and hence provides direction to the movement of reducing equivalents & to the production of ATP – to which it is coupled

Components of Respiratory chain• The components of the respiratory chain

are all present in the inner mitochondrial membrane as four protein – lipid respiratory chain complexes

• Cytochrome c is the only soluble cytochrome & together with ubiquinone seems to be a mobile component connecting the more fixed complexes

Summary In simple outline, ETC involves the removal

of hydrogen atoms from the oxidizable substrates; these hydrogen atoms enter the ETC, a system of membrane – bound complexes and each soon split to yield a proton and electron. These electrons then pass through a series of cytochromes, finally reacting with molecular oxygen and the protons that were released earlier, to form water

Electron carriers are Multi enzyme complexes• Complex I (NADH to Ubiquinone)• Also k/as NADH: ubiquinone reductase• Electron microscope reveals it as an L – shaped structure with

one arm in the membrane and the other extending into the matrix

• An enzyme with 42 polypeptide chains and an FMN – flavoprotein with about 6 Fe- S centers

• Complex I catalyses 2 simultaneous reactions1. - Exergonic transfer of a hydride ion (:H-)from NADH & a

proton from the matrix NADH + H+ + Q NAD+ + QH2

2. -Endergonic transfer of 4 protons from the matrix to the

intermembrane space Complex I – k/as proton pump driven by the energy of electron

transfer; where – protons move from one location (matrix which then becomes negatively charged) to the other (intermembrane space which becomes positively charged)

Structure of iron – sulfur center• These function as prosthetic groups which facilitate

electron transfer• Iron - sulfur protein - The iron is not present in heme

but is found in association with inorganic sulfur or the cysteine residues in the protein

• Rieske Iron - sulfur protein - One iron atom is coordinated to 2 Histidine residues instead of 2 cysteine residues

• All these centers however participate in one – electron transfer where the Fe –atom gets oxidized/ reduced

• At least 8 Fe – S proteins are involved in mitochondrial electron transfer

Ubiquinone • Lipid soluble bezoquinone with a long isoprenoid side chain • Complete reduction of ubiquinone requires 2 electrons and 2

protons ( a 2 step rxn) through semiquinone as an intermediate• As it carries both e- & protons , acts in coupling electron flow to

proton movement • It always acts at the junction between 2 e- donor and one electron

acceptor

O

O

CH 3O

CH 3CH 3O

(CH 2 CH C CH 2 )nH

CH 3

O H

O H

CH 3O

CH 3CH 3O

(CH 2 CH C CH 2 )nH

CH 3

e + 2 H +

c o e n zy m e Q

c o e n zy m e Q H 2

O

O

CH 3O

CH 3CH 3O

(CH 2 CH C CH 2 )nH

CH 3e

c o e n zy m e Q •

Complex II• Also called as Succinate:ubiquinone oxidoreductase

(Succinate to ubiquinone)• It is the only membrane bound enzyme (succinate

dehydrogenase) encountered in citric acid cycle • Structurally it is simpler than complex I with 2 types

of prosthetic groups & 4 different proteins • One of the protein is bound covalently to FAD & Fe- S

center with 4 Fe atoms• Electrons pass from succinate to FAD; through Fe – S

centers finally reaching ubiquinone

Oxidation of NADH & Succinate • NADH produced by CAC diffuses into the ETC where

the flavoprotein enzyme (NADH dehydrogenase) oxidizes it to NAD+

• The process involves transfer of a Hydride ion (which consists of hydrogen nucleus with 2 associated electrons) to the flavin enzyme (FMN) which accepts the proton to be FMNH2

• To be known – Hydride ions do not have independent existence – they just represent the moiety transferred in–biological reduction process

• Succinate Dehydrogenase flavoprotein having FAD as the coenzyme – links CAC directly to ETC

Fate of FMNH2 & FADH2

• FMNH2 & FADH2 are oxidized by enzymes that transfer the hydrogen atoms to a molecule of Ubiquinone (Q) thus forming Ubiquinol (QH2)

• Ubiquinone also accepts hydrogen atoms transferred from other molecules that have been oxidized by ETC ( - oxidation, glycerol – 3 – P)

Succinate dehydrogenase (fp)

Fatty acyl CoA dehydrogenase (fp)

Glycerol 3 phosphate dehydrogenase (fp)

NADH fp Dehydrogenase Fes

Q bc1

FeSQ

Matrix

Intermembrane space

Proton pump Proton pump

The Mechanism of Oxidation Phosphorylation

• Chemical coupling hypothesis• Conformational coupling hypothesis• Chemiosmotic hypothesis

ATP Synthase

Electron Transport Inhibitors

P/O Ratio

Uncouplers Disrupt the Coupling of Electron Transport

Reoxidation of Cytosolic NADH• The glycerol 3-phosphate shuttle

• The malate-aspartate shuttle