Redox Reactions in theMitochondrion

Module 0210101:

Molecular Biology and Biochemistry of the Cell

Lecture 12

Dale Sanders

17 February 2009

Objectives

1. how electrons flow spontaneously from NADH toO2, and the pathway they take in themitochondrial respiratory chain.

2. how “coupling sites” for the production of ATPcan be identified in the respiratory chain.

3. that ATP synthesis by respiratory chain“complexes” is powered by the redox potentialdifferences of reactions catalysed by thesecomplexes.

By the end of the lecture you should understand…

Reading

Voet & Voet (2004) Biochemistry (3rd Ed.) Chapter22 and especially pp. 802-820

All the topics today are well covered by the standard bigbiochemistry textbooks. An example is

Also useful for a more in-depth treatment is

Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3Chapter 5

FREE ENERGY, CARBOHYDRATECATABOLISM AND NADH

Oxidation of glucose yields a large amount of free energy

Glucose + 6 02 6 C02 + 6 H20 ΔG0’ = - 2850 kJ/mol

Much of this free energy is conserved by the reduction of NAD+ toNADH (nicotinamide adenine dinucleotide).

For each mol of glucose,

• 2 mol of NADH are produced in glycolysis (cytosolic)

• 2 mol from oxidation of pyruvate (mitochondrial)

• 6 mol of NADH in the citric acid cycle (mitochondrial).

Glucose (6C)

A Basic Summary of Production of ReducingEquivalents in Primary Metabolism

Gly-3-P (3C)

AcCoA

NADH

FADH2

NADH

NADH

NADH

PyruvateNADH

where –R = - ribose – P-P – ribose – adenine

NADH is subsequently oxidized in a reaction coupled to theproduction of ATP…

Reduction of NAD+

N

R

HH

CONH2 CONH2

+ H+ + 2e-

N

R

H

¨E'o = -0.32 V

+

Free energy from NADH oxidation [Recall : ΔE'0 = (E´0)ox – (E´0)red]

½ O2 + 2H+ + 2e- H2O E´0 = + 0.82 V

- NAD+ + H+ + 2e- NADH_____ E´0 = - 0.32 V

½ O2 + NADH + H+ NAD+ + H2O ΔE´0 = + 1.14 V

Since ΔG0’ = -nFΔE´0

ΔG0’ = - 2(96500)(1.14) = -220 kJ/mol

Since 10 mol NADH are produced, total free energy = -2200 kJ

[compare with 2850 kJ/mol glucose]

NADH NAD+

½ O2 H2O

ADP + Pi ATP

WHAT IS THE PATHWAY OF ELECTRONSFROM NADH O2?

Just like Glycolysis, this is not a single-step reaction:

Multiple steps allow conservation of free energy (asATP) at discrete points in pathway.

This reaction sequence accomplished by therespiratory chain which is an e- transport pathway.

The enzymes which catalyse the reactions of therespiratory chain are all membrane-bound: comprisea large fraction of the protein of the inner membrane.

http://www.biochemj.org/bj/361/0057/bj3610057.htm

Methods for investigating the components ofthe respiratory chain:1. Spectrophotometry: Many of the components (e.g.cytochromes) have characteristic absorption spectrawhich differ in the oxidized and reduced states:

e.g. cytochrome c absorption spectra

oxidized

reduced A handle onthe redoxstate oflight-absorbingcompounds

Methods……..2. Electron paramagnetic resonance (EPR)

Unpaired electrons detected by absorption of microwaveradiation in presence of varying magnetic field: results in

spectrum, characteristic for each component (quinones, Fe/S)

dA/dG

Magnetic field (H)

g value – a constant:

Diagnostic ofreactive group

http://www.mrc-dunn.cam.ac.uk/research/mitochondrial_complex_1/details.php

Methods…

3. Physical separation:

Weak detergents disrupt lipid-lipid interactions

Purification of macromolecular components(protein complexes) catalysing specific partialreactions in e- transport pathway

Reconstitute in lipid vesicles.

An Overview of the MitochondrialRespiratory Chain

4 Complexes, of which 3 are on NADH O2 pathway.

Complex II: Succinate dehydrogense.

NADH

NAD+

Complex I

Complex II

Complex III Complex IVUQ Cyt c

½O2

H2O

succinate fumarate

Identity of the Respiratory ChainComponents

1. Complex I (NADH – UQ oxidoreductase)

Catalyses transfer of 2e- from NADH to ubiquinone

NADH oxidized initially by a flavoprotein,

containing flavine mono-nucleotide as a prosthetic group.

o o

Electrons then passed to about 5 Fe/S centres(non-haem iron proteins)….

Identity of the Respiratory ChainComponents

Non-haem iron proteins:

Each Fe/S centre covalently linked to Cysresidues in protein

e.g. 4Fe:4S centreAlthoughseveral Fe,each centrereduced byjust 1 e-

2. Ubiqinone (Coenzyme Q10, CoQ10)

A lipid-soluble component,

present in great excess over other constituents of chain

CH3

quinone quinol

“Anti-fatigue, anti-diabetes,combating liver disease”

3. Complex III (UQ-cyt c oxidoreductase)

2 b-type cytochromes: b566 (-bL) and b562 (=bH)

1 Fe/S centre

cytochrome c1

Each cytochrome reduced by 1 e-

Cytochromes: a haem prosthetic group bound to aprotein.

The Fe atom coordinated in the porphyrin ring is reducedFe3+ Fe2+

4. Cytochrome c

The only component of the respiratory chain which isnot an integral part of the membrane.

Nevertheless, it is bound loosely to the OUTER sideof the inner membrane.

Artificial electron donor

Will accept e- from Ascorbate

5. Complex IV (cytochrome c oxidase)

cytochromes a, a3

2 copper atoms: CuA, CuB

Accepts 4 e- from cyt c (=4 separate turnovers of cyt c)

When fully reduced, can then reduce O2, together with4H+ 2H2O

Oxygen reduction on matrix side of membrane

Inhibitors: CN-, CO

6. Complex II (Succinate dehydrogenase)

Catalyses oxidation of succinate, reduction of UQ

• 3 Fe/S centres

• 1 flavine adenine dinucleotide (FAD)covalently bound to protein.

FAD: reduced in exactly the same way as FMN

o

Common Textbook Error

Succinate

http://jeb.biologists.org/cgi/content-nw/full/206/4/651/FIG1

Which Complexes Actually MakeATP?: Coupling Ratios

NADH

NAD+

½O2 + 2H+

H2OADP+ Pi

ATP

Measure O2 consumption by mitochondria in a closed chamberwith O2 electrode and observe

Mitosin Pi

buffer

malate

Pulse of ADP

ADP exhausted[O2]

time

Background respiration observed with respiratorysubstrate (malate).

Addition of ADP large increase in respiratory rate.When ADP is all phosphorylated, rate returns tobackground rate.

i.e. The e- transport and phosphorylation reactionsare COUPLED.

If a known amount of ADP is added, the amount ofO2 used during phosphorylation can be measured togive:

ADP: 0 ratio (P:O ratio)

P:O ratio is a measure of the number of ATP

molecules synthesized per pair of e- passing downthe respiratory chain.

P:O Ratios can be used to Identify theCoupling Sites in the Respiratory Chain

At which points of the resp. chain is thethermodynamically downhill flow of electrons coupled tosynthesis of ATP?

e- donor Active complexes Observed P:0

Malate/NADH I, III, IV ~3

Succinate II, III, IV ~2

Ascorbate IV ~1

• Conclude: Complexes I, III and IV are all coupling sites.

• However, no ATP is produced by Complex II.

4

Coupling Between Electron Transportand ATP Synthesis

E'o

(mV)

Summary

1. Free energy liberated during oxidation of glucoseto CO2 is largely conserved as NADH.

2. Oxidation of NADH occurs via a sequence of redoxreactions in the respiratory chain.

3. Complexes I, III and IV of the respiratory chaincatalyse reactions which are coupled to ATPproduction.