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Biological oxidation IRespiratory chain

• Metabolism

• Macroergic compound

• Redox in metabolism

• Respiratory chain

• Inhibitors of oxidative phosphorylation

Outline

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 energy-requiring processes of cells

“High energy” bonds

Phosphoanhydride bonds (formed by splitting out H2O

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 Pi

hydrolysis.

• Removal of Pi 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 Pi transfer.

• DG of phosphate hydrolysis from ATP is intermediate

among examples below.

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

by Pi transfer, e.g., from PEP.

Compound

DGo of phosphate hydrolysis (kJ/mol)

Phosphoenolpyruvate (PEP)

Phosphocreatine

Pyrophosphate

ATP (to ADP)

Glucose-6-phosphate

Glycerol-3-phosphate

• 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.

Some other

“high energy”

bonds:

• 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, PPi, 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): Fe2+ (reduced) Fe3+ (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 Pi.

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+ 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.

Oxidation of the coenzyme Q

Respiratory ChainAn Overview

• Electron Transport: Electrons carried byreduced 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 thesynthesis 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

27

Sequence of electron carriers in the respiratory chain

Complex I

proton pump

Complex II, does not

pump protons

Complex III

proton pump

Complex IV

proton pump

Coenzyme Q

electron shuttle

Cytochrome c

electron shuttle

Complex Name No. of

Proteins

Prosthetic Groups

Complex I NADH

Dehydrogenase

46 FMN, 9 Fe-S centers

Complex II Succinate-CoQ

Reductase

5 FAD, cyt b560, 3 Fe-S

centers

Complex III CoQ-cyt c

Reductase

11 cyt bH, cyt bL, cyt c1,

Fe-SRieske

Complex IV Cytochrome

Oxidase

13 cyt a, cyt a3, CuA, CuB

Complexes of Respiratory chain

Complex INADH-CoQ Reductase

• Electron transfer from

NADH to CoQ

• Path: NADH FMN

Fe-S UQ FeS

UQ

• Four H+ transported

out per 2 e-

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., Fe3+ ).

Complex IISuccinate-CoQ Reductase

• aka succinate dehydrogenase (from TCA cycle!)

• aka flavoprotein 2 (FP2) - FAD covalently bound

• four subunits, including 2 Fe-S proteins

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

• Path: succinate FADH2 2Fe2+ UQH2

• Net reaction: succinate + UQ fumarate + UQH2

Complex IIICoQ-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

• UQH2 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 IVCytochrome c Oxidase

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

reduction of O2 to produce 2H2O

• Oxygen is thus the terminal acceptor of

electrons in the electron transport pathway -

the end!

• Cytochrome c oxidase utilizes 2 hemes (a and

a3) 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

2005-2006

Peter Mitchell• Proposed chemiosmotic hypothesis

– revolutionary idea at the time

1961 | 1978

1920-1992

proton motive force

ATP Synthase

ATP synthase

subunit

c ring subunit

subunit b subunit

F1 subunit has 5 types of

polypeptide chains

(3, b3, , , ), displays

ATPase activity

and b are members of

P-loop family

F0 contains the proton channel

ring of 10-14 c subunits„a‟ subunit binds

to outside of ring

Exterior column

has 1 a subunit

2 b subunits, and

the subunit

Moving unit (rotor) is c ring and

Remainder is stationary (stator)

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 - F1Fo 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 Fo "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 (FADH2), 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 (Fe3+) of a3

• Oligomycin are ATP synthase

inhibitors

UncouplersUncoupling 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 uncoupler-

stimulated 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.

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

Inhibitors of respiratory chain