chapter 6 biological oxidation

Chapter 6

Biological Oxidation

The biochemistry and molecular biology department of CMU

Biological oxidation is the proc

ess in which substances (carb

ohydrate, Lipid, AAs) are oxidiz

ed in living organism.

Oxidation types:

Dehydrogenation

Electron lost

Oxygenation

Nature of biological oxidation ：1. 37 , pH 7.4, enzymatic reaction.℃2. Energy released gradually.

3. Formation of H2O.

4. Formation of CO2 by decarboxylation.

CO2

ATP ADP + Pi1/2 O2

H2O

acetyl CoA CoASH

G glycerol, FAs AAs

glycogen lipids proteinsfirst

phase

secondphase

thirdphaseoxidative

phosphorylation

Pyr

TCAC

cytosol

Mit.

2H

2H

General situation of biological oxidation

§1 Respiratory Chain

§2 Oxidative Phosphorylation

§3 ATP

§4 Shuttle Systems

§ 1 Respiratory Chain

A chain in the mitochondria con

sists of a number of redox carriers f

or transferring hydrogens removed f

rom the substrate to oxygen to form

water. The chain is termed a respirat

ory chain, also called electron trans

port chain (ETC).

Composition of Respiratory Chain Complexes

Complexes NameProsthetic Groups

Complex I NADH-CoQ Reductase

FMN, Fe-S

Complex II Succinate-CoQ Reductase

FAD, Fe-S

Complex III CoQ-Cyt c Reductase

iron-porphyrine

Fe-S

Complex IV Cytochrome Oxidase

iron-porphyrine Cu

Position of Respiratory Chain Complexes

Order of Respiratory Chain Complexes

NADH

succinate

FMN£¨Fe-S£©

FAD£¨Fe-S£©

Cyt b, £¨Fe-S£©CoQ Cyt aa3 O2Cyt ccompex I

compex II

compex III compex IV

c1

Composition of ETC

　　 Hydrogen carrier ： NAD +

　　　　　　　 FMN

　　　　　　　 FAD

　　　　　　　 CoQ

　　 electron carrier ： Fe-S 　　　　　　　　　　　　　 Cyt

• NAD+/NADH (Nicotinamide Adenine Dinucleotide, Co )Ⅰ

• NADP+/NADPH (Nicotinamide Adenine Dinucleotide Phosphate, Co

)Ⅱ

The nicotinamide is the vitamin PP.

1. Nicotinamide coenzymes

NAD+

O

H H

OH OH

H H

CH2 O O

OHOH

HHH

CH2

H

OP

OH

O

N

N

N

N

NH2

N

CONH2

O P

OH

O

NADP+

O

H H

OH OH

H H

CH2 O O

OOH

HHH

CH2

H

OP

OH

O

N

N

N

N

NH2

N

CONH2

O P

OH

O

PO

OH

OH

N

H

CONH2

R

+ H + H + + e

N

H

CONH2

R

H

+ H +

NAD +/NADP + NADH/NADPH

FMN: Flavin Mononucleotide

FAD: Flavin Adenine Dinucleotide

They contain the riboflavin (Vit B2).

2. Flavin prosthetic groups

FMN

CH2 OHO P

OH

OC

C

H OH

C

H OH

H OH

CH H

NH

N

N

N O

O

H3C

H3C1

45

8 9

10Isoalloxazine

ribitol

FAD

CH2 O O

OHOH

HHH

CH2

H

OP

OH

O

N

N

N

N

NH2

O P

OH

OC

C

H OH

C

H OH

H OH

CH H

NH

N

N

N O

O

H3C

H3C1

45

8 9

10

R

NH

N

N

N O

O

H3C

H3C

FMN/FAD

1

45

8 9

10

R

NH

NH

HN

N O

O

H3C

H3C1

45

8 9

10+ 2H

FMNH2/FADH2

NAD+ is a coenzyme, that

reversibly binds to enzymes.

FAD is a prosthetic group, that

remains tightly bound at the active

site of an enzyme.

Iron-sulfur centers (Iron-sulfur

protein, Fe-S) are prosthetic groups

containing 2, 3, 4 or 8 nonheme iron

atoms complexed to elemental and

cysteine S.

3. Fe-S

Fe

Fe

S

S

S

Fe

Fe

S

S

S

SS

Cys

Cys

Cys

Cys

S

Fe

S

Fe

S

S

S

S

Cys

CysCys

Cys

Iron-sulfur centers

2 Fe iron-sulfur center of ferredoxin.

2 Fe colored orange; elemental & Cys S yellow.

Different types of iron-sulfur centers

Iron-sulfur centers transfer only

one electron.

Fe3+ + e- Fe2+

Coenzyme Q (CoQ, ubiquinone) is very hydrophobic. It dissolves in the membrane.

Coenzyme Q functions as a mobile e- carrier within the mitochondrial inner membrane.

4. CoQ

O

O

H3CO

H3CO

CH3

(CH2 CH

C

CH3

CH2)nH

CoQ

isoprene

H2C C C CH2

CH3

H

O

O

CH3O

CH3CH3O

R

OH

OH

CH3O

CH3CH3O

R

+ 2H

ubiquinone ubiquinol

Cytochromes (Cyt) are proteins with heme prosthetic groups. They absorb light at characteristic wavelengths.

Hemes in the 3 classes of cytochrome (a, b, c) differ slightly in substituents on the porphyrin ring system.

5. Cytochromes

Hemes a & a3 are often referred to as cytochromes aa3.

Cytochrome c is a small, water-soluble protein with a single heme group.

N

N

N

N

CH3 CH

CH2

CH3

CH CH2

CH2

CH2

COO

CH3

HC

CH2CH2 OOC

Fe

O

Heme b

N

N

N

N

CH3 HC

CH3

S CH2

CH3

CH S CH2

CH3

CH2

CH2

COO

CH3

H3C

CH2CH2 OOC

protein

protein

Fe

Heme c

Heme c

Cytochrome c

Lys13 Lys 72

heme

complex IV

cyt. c

N

N

N

N

CH3 HC

CH2

CH3

CH CH2

CH2

CH2

COO

CH3

HC

CH2CH2 OOC

Fe

OH

CH2 CH C CH2

CH3

3 H

O

Heme a

The heme iron can undergo

an electron transition between

ferric and ferrous states:

Fe3+ + e- Fe2+

The sequence of the components in the respiratory chain has been deduced in several ways.

1. Inhibitor.

2. O2 is suddenly introduced into the system.

3. Standard redox potential.

Inhibitor

Standard reduction potentials for respiratory chain and related electron carriers

1. NADH respiratory chain ：

2. Succinate respiratory chain ： c1 c aa3 O2

FAD£¨ Fe-S£©

succinate CoQ Cyt b

NADH FMN£¨Fe-S£©

c1 c aa3 O2

FAD£¨Fe-S£©

succinate¦Á-glycerophosphate

CoQ Cyt b

malate¦Â-hydroxy fatty acyl CoA¦Â-hydroxybutyrateisocitriteGlu

FAD

lipoic acid

pyruvate¦Á-ketoglutarate

FAD

fatty acyl CoA

§2

Oxidative Phosphorylation

Oxidative phosphorylation:

The phosphorylation of ADP t

o ATP coupled to electron transfer

from a substrate to molecular oxy

gen.

Substrate level phosphorylation ：

Phosphorylation of ADP or GD

P to ATP or GTP coupled to the deh

ydrogenation of an organic substra

te.

Coupled site:

1. P/O ratio is the number of

inorganic phosphates

incorporated into ATP per

oxygen atom consumed.

(number of ATP / 2H)

P/O ratio of some substrate

substratescomponent

sP/O ratio

Number of ATP

-hydroxybutyrate NAD+→O2 2.4~2.8 3

succinate FAD →O2 1.7 2

Vit C Cyt c→O2 0.88 1

Cyt c Cyt aa3→O20.61~0.6

81

∆E0'E0'

NADH FMN£¨ Fe-S£©

CoQ Cyt b Cyt c1 Cyt c Cyt aa3 O2

FAD£¨ Fe-S£©

succinate

energyADP + Pi ATP

energyADP + Pi ATP

energyADP + Pi ATP

-0.32 -0.22 +0.04 +0.08 +0.23 +0.25 +0.29 +0.82

0.36V 0.21V 0.53V

69.5kJ/mol 40.5kJ/mol 102.3kJ/mol∆G0'

-hydroxybutyrate

succinate

Vit C

Three Coupled sites

① NADH → CoQ

② CoQ → Cyt c

③ Cyt aa3 → O2

• NADH ETC has 3 coupled site ， P/O ratio =3,

3mol ATP / 2H.

• Succinate ETC has 2 coupled site ， P/O ratio =2 ， 2mol ATP / 2H.

Coupled mechanism:

1. Chemiosmotic hypothesis

The chemiosmotic hypothesis is the concept that a proton concentration gradient serves as the energy reservoir for driving ATP formation and was originally formulated by Peter Mitchell in the early 1960s .

Respiratory chain is in cristae of the inner membrane.

inner membranecristae

outer membrane

Intermembrane space

matrix

Spontaneous electron flow

through each of complexes I, III, &

IV is coupled to H+ ejection from the

matrix. 

A total of 10 H+ are ejected from the mitochondrial matrix per 2 e- transferred from NADH to oxygen via the respiratory chain.

Matrix

NADH + H+ NAD+

2H+ + ½ O2 H2O

2 e – – I Q III IV

+ +

4H+ 4H+ 2H+ Intermembrane Space

cyt c

2. ATP synthase (complex V )

ATP synthase, embedded in cristae of the inner mitochondrial membrane, includes:

F1 catalytic subunit, made of 5 kinds of polypeptides α3β3γδε.

F0 complex of integral membrane proteins mediates proton transport.

Looking down at the membrane, they are arranged as a ring of alternating α & β subunits.

F1 in cross

section

ADP + Pi ATP

ATP

ATP ADP

+ Pi ATP ADP

+ Pi

ATP open tight

binding

loose binding

The mechanism for ATP synthase

Matrix

NADH + H+ NAD+ 2H+

+ ½ O2 H2O

2 e – – I Q III IV

+ +

4H+ 4H+ 2H+ Intermembrane Space

cyt c 3H+

F1

Fo

ADP + Pi ATP

3H+ 3H+

Transport of ATP, ADP, & Pi

summary

2.5 ~P bonds synthesized during oxidation of NADH produced in the matrix. (10 H+ pumped; 4 H+ used up per ATP).

1.5 ~P bonds synthesized during oxidation of FADH2 produced in the

matrix.

Regulation of Oxidative

Phosphorylation

1. The factors affecting ETC

A. Inhibitors of ETC

NADHFMN

£¨Fe-S£© CoQ Cyt b Cyt c1 Cyt c Cyt aa3 O2

FAD£¨Fe-S£©

succinate

Rotenone, Amytal, piericidin A

dimercaptopropanol Antimycin A

CN-, N-3,

CO, H2S

B. Uncoupling agent

A compound that disrupts the

usual tight coupling between elect

ron transport and phosphorylation

of ADP.

Uncoupling reagents dissolve in the membrane and function as carriers for H+.

OH

NO2

NO2

2,4-dinitrophenol

Uncouplers block oxidative phosphorylation by

dissipating the H+ electrochemical gradient.

Matrix

NADH + H+ NAD+ 2H+

+ ½ O2 H2O

2 e I Q III IV

4H+ 4H+ 2H+ H+ Intermembrane Space

cyt c

uncoupler

Endogenous

Uncouplers E

nable Organi

sms To Gene

rate Heat.

C. Inhibiter of oxidative phosphoryl

ation

A compound that Inhibits electron

transport and phosphorylation of A

DP.

Oligomycin

2. ADP

[ADP]↑→ Oxidative phosphorylation↑

[ADP]↓→ Oxidative phosphorylation↓

pyruvate

ADP

succinate

Mt.

rotenone

oligomycin

antimycin A ( CN-)

time

DNP

Influence of different substrates and inhibitors on quantity consuming oxygen

3. Thyroxine

Na+,K+-ATPase →ATP catabolism → ADP↑ → Oxidative phosphorylation↑ → formation of ATP↑. Formation and catabolism of ATP↑ ， consuming of O2↑, Generation of Heat↑.

4. mtDNA mutation

Free radical → mutation of DNA → inhibition of Oxidative phosphorylation → energy↓

§3 ATP

N

N

N

N9

NH2

O

OHOH

HH H

CH2

H1'

2'

glycosidicbondOP

O-

O

O

PO

O-

O

P

O-

-O

O¦Á¦Â¦Ã

ester bond

Adenosine triphosphate, ATP

α bond G△ 0′ ＝ － 14.3KJ

β bond G△ 0′ ＝ － 32.2KJ

γ bond G△ 0′ ＝ － 30.5KJ

>30KJ （ 7kcal ） -high energy bond

s

Compound Go' of phosphate hydrolysis, kJ/mol

Phosphoenolpyruvate (PEP)

Phosphocreatine

Pyrophosphate

ATP (to ADP)

Glucose-6-phosphate

Glycerol-3-phosphate

ΔG0' of phosphate hydrolysis of some compound

Examples of high-energy compounds

Type of bond General formula Example

ΔG0'kJ/mol

Phospho-guanidines

Creatine phosphate 43.9

Enol phosphates PEP 61.9

Phosphoric-carboxylic

1,3-bisphospho-

glycerate41.8

Phosphoric acid

anhydrides

ATP, GTP, UTP, CTP 30 .5

Thiol estersAcetyl CoA,

Acyl CoA31.4

R C O

CH2

~ PO3H2

R C N

NH

H

~ PO3H2

R C O

O~ PO3H2

P O

O

~P OHOH

O

OH

R C

O

~ SCoA

Potentially, 2 ~P bonds can be cleaved, as 2 phosphates are released by hydrolysis from ATP.

AMP~P~P AMP~P + Pi

(ATP ADP + Pi)

AMP~P~P AMP + P~P

(ATP AMP + PPi)

P~P 2 Pi

(PPi 2Pi)

Adenylate Kinase:

  2 ADP ATP + AMP

Nucleoside Diphosphate Kinase catalyzes

reversible reactions such as:

ATP + GDP ADP + GTP,

ATP + UDP ADP + UTP, etc.

Phosphocreatine is used in nerve and muscle for storage of ~P bonds.

creatinephosphate

+ ADP

creatine

+ ATP

creatinekinase

COOH

CH2

N

C

H3C

NH2

NH

COOH

CH2

N

C

H3C

N

NH

H

P

Production and application of ATP

creatinephophate

creatine

ATP

~P

ADP

~P

oxidativephosphorylation

Substrate level phosphorylation

mechanism energyosmotic energychemical energyelectric energyheat energy

§4 Shuttle Systems

Permeable to ：

Pyr, succinate, α-ketoglutarate, malate, citrite, Glu etc.

Impermeable to ：

H+, NADH, NADPH, OAA etc.

The inner membrane of mitochondria is quite impermeable to some molecules and ions.

• Oxidation of extramitochondrial NA

DH is mediated by shuttle systems.

- Glycerophosphate shuttle system

Brain, skeletal muscle

Malate-asparate shuttle ( heart , liver )

NAD+

NADH+H+

Malate

OAA

Malate

OAAGluGlu

Asp Asp¦Á-KG ¦Á-KG

NAD+

NADH+H+

ETC

3ATP

MDH

transaminasetransaminase

MDH

cytosol matrix