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Biological oxidation I

Respiratory chain


Outline

• Metabolism

• Macroergic compound

• Redox in metabolism

• Respiratory chain

• Inhibitors of oxidative phosphorylation


Metabolism

• Metabolism consists of catabolism and

anabolism

• 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

hydrolysis.

• 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-

phosphate.


• 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.

Compound

Phosphoenolpyruvate (PEP)

Phosphocreatine

Pyrophosphate

ATP (to ADP)

Glucose-6-phosphate

Glycerol-3-phosphate

DG o

of phosphate hydrolysis (kJ/mol)


Some other

“high energy”

bonds:

• 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

b-mercaptoethylamine,

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

b-mercaptoethylamine.


“High energy” (macroergic) compounds

exemplifying the following roles:

• Energy transfer or storage

ATP, PP i , polyphosphate, creatinephosphate

• Group transfer

ATP, Coenzyme A

• Transient signal

cAMP


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

• 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.

NADP + /NADPH is

similar except for P i .

NADPH is e donor in

synthetic pathways.


NAD + /NADH

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

enzyme.


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

complexes

• 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

27


Complexes of Respiratory chain

Complex Name No. of

Proteins

Prosthetic Groups

Complex I

Complex II

NADH

Dehydrogenase

Succinate-CoQ

Reductase

46 FMN, 9 Fe-S centers

5 FAD, cyt b 560

, 3 Fe-S

centers

Complex III CoQ-cyt c

Reductase

Complex IV Cytochrome

Oxidase

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

NADH to CoQ

• Path: NADH FMN

Fe-S UQ FeS

UQ

• 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

carrier

• cyt c is a water-soluble electron

carrier


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

2005-2006

1920-1992


ATP Synthase


subunit

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

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

gradients.


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

membrane

• 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

Phosphorylation

• 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

inhibitors


Uncouplers

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

proton

• They shuttle back and forth

across the membrane, carrying

protons to dissipate the

gradient


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

uncouplers.

2. Phosphorylation inhibitors (e.g. oligomycin)

abolish the burst of oxygen consumption after

adding ADP, but have no effect on uncouplerstimulated

respiration.


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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