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Biological oxidation I - TOP Recommended Websites

Biological oxidation I

Respiratory chain


• Metabolism

• Macroergic compound

• Redox in metabolism

• Respiratory chain

• Inhibitors of oxidative phosphorylation


• Metabolism consists of catabolism and


• Catabolism: degradative pathways

– Usually energy-yielding!

• Anabolism: biosynthetic pathways

– energy-requiring!

The ATP Cycle

• ATP is the energy currency of cells

• In phototrophs, light energy is transformed

into the light energy of ATP

• In heterotrophs, catabolism produces ATP,

which drives activities of cells

• ATP cycle carries energy from

photosynthesis or catabolism to the energyrequiring

processes of cells

“High energy” bonds

Phosphoanhydride bonds (formed by splitting out H 2 O

between 2 phosphoric acids or between carboxylic and

phosphoric acids) have a large negative DG of hydrolysis.

Phosphoanhydride linkages are said to be "high energy"

bonds. Bond energy is not high, just DG of hydrolysis.

"High energy" bonds are represented by the "~" symbol.

~P represents a phosphate group with a large negative DG

of hydrolysis.

• Phosphocreatine (creatine phosphate), another

compound with a "high energy" phosphate linkage, is

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

• Phosphocreatine is produced when ATP levels are high.

• When ATP is depleted during exercise in muscle,

phosphate is transferred from phosphocreatine to ADP,

to replenish ATP.

• Phosphoenolpyruvate (PEP), involved in ATP

synthesis in Glycolysis, has a very high DG of P i


• Removal of P i from ester linkage in PEP is spontaneous

because the enol spontaneously converts to a ketone.

• The ester linkage in PEP is an exception.

Other examples of phosphate esters with low but

negative DG of hydrolysis:

• the linkage between phosphate and a hydroxyl

group in glucose-6-phosphate or glycerol-3-


• ATP has special roles in energy coupling and P i transfer.

• DG of phosphate hydrolysis from ATP is intermediate

among examples below.

• ATP can thus act as a P i donor, and ATP can be synthesized

by P i transfer, e.g., from PEP.


Phosphoenolpyruvate (PEP)



ATP (to ADP)



DG o

of phosphate hydrolysis (kJ/mol)

Some other

“high energy”


• A thioester forms between a carboxylic acid and a thiol

(SH), e.g., the thiol of coenzyme A (abbreviated CoA-SH).

• Thioesters are ~ linkages. In contrast to phosphate esters,

thioesters have a large negative DG of hydrolysis.

• The thiol of coenzyme A can react with a carboxyl

group of acetic acid (yielding acetyl-CoA) or a fatty

acid (yielding fatty acyl-CoA).

• The spontaneity of thioester cleavage is essential to the

role of coenzyme A as an acyl group carrier.

• Like ATP, CoA has a high group transfer potential.

Coenzyme A includes


in amide linkage to the

carboxyl group of the B

vitamin pantothenate.

The hydroxyl of

pantothenate is in ester

linkage to a phosphate

of ADP-3'-phosphate.

The functional group is

the thiol (SH) of


“High energy” (macroergic) compounds

exemplifying the following roles:

• Energy transfer or storage

ATP, PP i , polyphosphate, creatinephosphate

• Group transfer

ATP, Coenzyme A

• Transient signal


Oxidation and reduction

• Oxidation of an iron atom involves loss of an electron (to

an acceptor): Fe 2+ (reduced) Fe 3+ (oxidized) + e -

• Since electrons in a C-O bond are associated more with

O, increased oxidation of a C atom means increased

number of C-O bonds.

• Oxidation of C is spontaneous.

Increasing oxidation number of C

Redox in Metabolism

• NAD + collects electrons released in


• Catabolism is oxidative - substrates lose

reducing equivalents, usually H + ions

• Anabolism is reductive – NAD(P)H

provides the reducing power (electrons) for

anabolic processes

NAD + , Nicotinamide

Adenine Dinucleotide,

is an electron acceptor

in catabolic pathways.

The nicotinamide ring,

derived from the

vitamin niacin, accepts

2 e - and 1 H + (a

hydride) in going to the

reduced state, NADH.


similar except for P i .

NADPH is e donor in

synthetic pathways.


The electron transfer reaction may be

summarized as :

NAD + + 2e + H + NADH.

It may also be written as:

NAD + + 2e + 2H + NADH + H +

FAD (Flavin Adenine Dinucleotide), derived from the

vitamin riboflavin, functions as an e acceptor. The

dimethylisoalloxazine ring undergoes reduction/oxidation.

FAD accepts 2 e - + 2 H + in going to its reduced state:

FAD + 2 e - + 2 H + FADH 2

• NAD + is a coenzyme, that reversibly

binds to enzymes.

• FAD is a prosthetic group, that remains

tightly bound at the active site of an


Oxidation of the coenzyme Q

Respiratory Chain

An Overview

• Electron Transport: Electrons carried by

reduced coenzymes are passed through a

chain of proteins and coenzymes to drive

the generation of a proton gradient across

the inner mitochondrial membrane

• Oxidative Phosphorylation: The proton

gradient runs downhill to drive the

synthesis of ATP

• It all happens in or at the inner

mitochondrial membrane

Electron Transport

• Four protein complexes in the inner

mitochondrial membrane

• A lipid soluble coenzyme (UQ, CoQ) and a water

soluble protein (cyt c) shuttle between protein


• Electrons generally fall in energy through the

chain - from complexes I and II to complex IV

Sequence of electron carriers in the respiratory chain

Coenzyme Q

electron shuttle

Complex I

proton pump

Complex II, does not

pump protons

Cytochrome c

electron shuttle

Complex III

proton pump

Complex IV

proton pump


Complexes of Respiratory chain

Complex Name No. of


Prosthetic Groups

Complex I

Complex II





46 FMN, 9 Fe-S centers

5 FAD, cyt b 560

, 3 Fe-S


Complex III CoQ-cyt c


Complex IV Cytochrome


11 cyt b H

, cyt b L

, cyt c 1


Fe-S Rieske

13 cyt a, cyt a 3

, Cu A

, Cu B

• Electron transfer from


• Path: NADH FMN



• Four H + transported

out per 2 e-

Complex I

NADH-CoQ Reductase

Role of FMN: Since it can accept/donate either 1 or 2 e - ,

FMN has an important role in mediating electron transfer

between carriers that transfer 2 e - (e.g., NADH) and

carriers that can only accept 1 e - (e.g., Fe 3+ ).

Complex II

Succinate-CoQ Reductase

• aka succinate dehydrogenase (from TCA cycle!)

• aka flavoprotein 2 (FP 2 ) - FAD covalently bound

• four subunits, including 2 Fe-S proteins

• Three types of Fe-S cluster: 4Fe-4S, 3Fe-4S, 2Fe-2S

• Path: succinate FADH 2 2Fe 2+ UQH 2

• Net reaction: succinate + UQ fumarate + UQH 2

Complex III

CoQ-Cytochrome c Reductase

• CoQ passes electrons to cyt c (and

pumps H + ) in a unique redox cycle

known as the Q cycle

• The principal transmembrane protein

in complex III is the b cytochrome

• Cytochromes, like Fe in Fe-S clusters,

are one- electron transfer agents

• UQH 2 is a lipid-soluble electron


• cyt c is a water-soluble electron


Heme is a prosthetic group of cytochromes. Heme contains an iron

atom embedded in a porphyrin ring system. The Fe is bonded to 4 N

atoms of the porphyrin ring. Hemes in the three classes of cytochrome

(a, b, c) differ slightly in substituents on the porphyrin ring system. A

common feature is two propionate side-chains.

Complex IV

Cytochrome c Oxidase

• Electrons from cyt c are used in a four-electron

reduction of O 2 to produce 2H 2 O

• Oxygen is thus the terminal acceptor of

electrons in the electron transport pathway -

the end!

• Cytochrome c oxidase utilizes 2 hemes (a and

a 3 ) and 2 copper sites

• Complex IV also transports H +

Coupling e - Transport and

Oxidative Phosphorylation

This coupling was a mystery for many years

• Many biochemists squandered careers searching

for the elusive "high energy intermediate"

• Peter Mitchell proposed a novel idea - a proton

gradient across the inner membrane could be

used to drive ATP synthesis

• Mitchell was ridiculed, but the chemiosmotic

hypothesis eventually won him a Nobel prize

Peter Mitchell

• Proposed chemiosmotic hypothesis

– revolutionary idea at the time

1961 | 1978

proton motive force



ATP Synthase


ATP synthase

Moving unit (rotor) is c ring and

Remainder is stationary (stator)

c ring subunit

„a‟ subunit binds

to outside of ring

Exterior column

has 1 a subunit

2 b subunits, and

the subunit


F 0 contains the proton channel

ring of 10-14 c subunits

F 1 subunit has 5 types of

polypeptide chains

( 3 , b 3 , , , ), displays

ATPase activity

b subunit

and b are members of

P-loop family

The Chemiosmotic Theory of oxidative phosphorylation,

for which Peter Mitchell received the Nobel prize:

Coupling of ATP synthesis to respiration is indirect,

via a H + electrochemical gradient.

Chemiosmotic theory - respiration:

Spontaneous e transfer through complexes I, III, & IV is

coupled to non-spontaneous H + ejection from the matrix.

H + ejection creates a membrane potential (DY, negative

in matrix) and a pH gradient (DpH, alkaline in matrix).

Chemiosmotic theory - F 1

F o

ATP synthase:

Non-spontaneous ATP synthesis is coupled to spontaneous

H + transport into the matrix. The pH and electrical gradients

created by respiration are the driving force for H + uptake.

H + return to the matrix via F o

"uses up" pH and electrical


ATP-ADP Translocase

ATP must be transported out of the mitochondria

• ATP out, ADP in - through a "translocase"

• ATP movement out is favored because the

cytosol is "+" relative to the "-" matrix

• But ATP out and ADP in is net movement of a

negative charge out - equivalent to a H + going in

• So every ATP transported out costs one H +

• One ATP synthesis costs about 3 H +

• Thus, making and exporting 1 ATP = 4H +

What is the P/O Ratio?

i.e., How many ATP made per electron pair through

the chain?

• e - transport chain yields 10 H + pumped out per

electron pair from NADH to oxygen

• 4 H + flow back into matrix per ATP to cytosol

• 10/4 = 2.5 for electrons entering as NADH

• For electrons entering as succinate (FADH 2 ), about

6 H + pumped per electron pair to oxygen

• 6/4 = 1.5 for electrons entering as succinate

Shuttle Systems for e -

Most NADH used in electron transport is cytosolic

and NADH doesn't cross the inner mitochondrial


• What to do?

• "Shuttle systems" effect electron movement

without actually carrying NADH

• Glycerophosphate shuttle stores electrons in

glycerol-3-P, which transfers electrons to FAD

• Malate-aspartate shuttle uses malate to carry

electrons across the membrane

Respiratory chain =

oxidative phosphoryltion

+ electron transport

Inhibitors of Oxidative


• Rotenone inhibits Complex I - and

helps natives of the Amazon rain forest

catch fish!

• Cyanide, azide and CO inhibit

Complex IV, binding tightly to the

ferric form (Fe 3+ ) of a 3

• Oligomycin are ATP synthase



Uncoupling e- transport and

oxidative phosphorylation

• Uncouplers disrupt the tight

coupling between electron

transport and oxidative

phosphorylation by dissipating

the proton gradient

• Uncouplers are hydrophobic

molecules with a dissociable


• They shuttle back and forth

across the membrane, carrying

protons to dissipate the


Uncouplers and Inhibitors

There are six distinct types of poison which may

affect mitochondrial function:

1. Respiratory chain inhibitors (e.g. cyanide,

antimycin, rotenone and TTFA) block

respiration in the presence of either ADP or


2. Phosphorylation inhibitors (e.g. oligomycin)

abolish the burst of oxygen consumption after

adding ADP, but have no effect on uncouplerstimulated


3. Uncoupling agents (e.g. dinitrophenol, CCCP, FCCP)

abolish the obligatory linkage between the respiratory

chain and the phosphorylation system which is observed

with intact mitochondria.

4. Transport inhibitors (e.g. atractyloside, bongkrekic

acid, NEM) either prevent the export of ATP, or the

import of raw materials across the the mitochondrial

inner membrane.

5. Ionophores (e.g. valinomycin, nigericin) make the inner

membrane permeable to compounds which are ordinarily

unable to cross.

6. Krebs cycle inhibitors (e.g. arsenite, aminooxyacetate)

which block one or more of the TCA cycle enzymes, or

an ancillary reation.

Inhibitors of respiratory chain

Name Function Site of action

retenone e transport inhibitor Complex I

amytal e transport inhibitor Complex I

antimycin A e transport inhibitor Complex III

cyanide e transport inhibitor Complex IV

carbon monoxide e transport inhibitor Complex IV

azide e transport inhibitor Complex IV

2,4-initrophenol uncoupling agent transmembrane H+ carrier

pentachlorophenol uncoupling agent transmembrane H+ carrier

oligomycin inhibits ATP-ase OSCP protein

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