Chapter 10

n Electron Transport – electron transfer to oxygen

n Oxidative Phosphorylation –conversion of ADP to ATP

n Chemiosmotic coupling –drives synthesis of ATP

Aerobic Metabolism II: Electron Transport and Oxidative Phosphorylation

Overview

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.1: Electron TransportSeries of electron carriers in order

of increasing electron affinity§Inner mitochondrial membrane

NADH/FADH2 -> Co-Q -> cytochromes -> O2§ Glucose à CO2 + water

§Aerobic respiration couples electron transfer ultimately to ATP synthesis

§Protons pumped (H+) creates pH gradient

§Drives synthesis of ATP

§Chemiosmotic coupling – results from stored potential energy, basis for coupling between oxidation & phosphorylation

Figure 10.1 The Electron Transport Chain

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.1: Electron Transport§NADH à E-FMN§E-FMN à Fe clusters Complex I§Fe clusters à Co-enzyme Q

§Co-enzyme Q à Cytochrome b (Q cycle)

§Cytochrome b à Fe clusters§Fe clusters à Cytochrome C Complex III

§Cytochrome C à Cytochrome A§Cytochrome A à oxygen Complex IV

Section 10.1: Electron Transport

§Complex I - transfer of electrons fromNADH to ubiquinone (CoQ) CoQH2§NADH dehydrogenase complex

§Large complex - >20 subunits §1 molecule FMN, 7 Fe clusters

§Flavin mononucleotide (FMN) oxides NADH à FMNH2

Figure 10.2 Two Iron-Sulfur Clusters

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

NADH + H+ E-FMN àNAD+ + E-FMNH2 à

Feox à Fered à CoQ à CoQH2

Section 10.1: Electron Transport

§Electrons transfer: NADH to FMN àFMNH2

§FMNH2 à iron sulfur centers§Iron/sulfur centers à CoQ§Movement of protons from matrix

to intermembrane space

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Figure 10.4 Electron Movement throughComplex I of the Electron Transport Chain

Section 10.1: Electron Transport- transfers electrons from succinate from CAC to UQ

§Succinate dehydrogenase complex§Four subunits (ShdA-D) – ShdA - succinate binding site;

ShdB - 3 iron-sulfur clusters; ShdC & D integral membrane proteins

§Located in inner mitochondrial membrane§Does not translocate protons

Figure 10.5 Path of Electrons from Succinate, Glycerol-3-Phosphate, and Fatty Acids to UQ

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.1: Electron Transport

- electrons from reduced UQ (UQH2) to cytochrome c

Cytochrome bc1 complex§Cytochromes - proteins with heme

prosthetic group§cyt b, cyt c, several iron/sulfur

proteins§Electrons change oxidation state of

heme iron (reduced Fe2+ to oxidized Fe3+)

Figure 10.6 Structure of Cytochrome c

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.1: Electron Transport

- transfer of electrons through complex III§Cytochrome c is a water-soluble mobile electron carrier on outer face

of the inner membrane§Two molecules of cyt c per one molecule of CoQ

§Cyt c carries e-, H+ leaves matrixCoQH2 = 2cyt cox (Fe+3) = 2H+

matrix à CoQ + 2cyt cred(Fe+2) + 4H+cytosol

Figure 10.7 Electron Transport throughComplex III

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.1: Electron Transport

§ Four electrons & four protons are passed to O2 to form H2O2Cyt c[Fe(II)] + 2H+ + 1/2O2 ---> 2 Cyt c[Fe(III)] + H2O

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§ATP - allosteric inhibitor of cytochrome oxidase§Binds to complex IV and cyt c

- four electron reduction of O2 to H2O

§cytochrome oxidase§Contains cytochrome a, a3, 3 copper

ions§CuA-CuA accepts electrons, passes to

cyt a, àcyt a3 à CuB

Section 10.1: Electron Transport

Section 10.1: Electron Transport

§NADH oxidation -substantial energy release§Used to pump protons into

intermembrane space§Establishes a proton gradient

§2.5 molecules ATP per NADH§1.5 molecules ATP per FADH2

Figure 10.9 Energy Relationships in the Electron Transport Chain

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.1: Electron Transport Inhibitors

§When electron transport is inhibited, O2 consumption isreduced or eliminated

§Antimycin A inhibits cyt b in Complex III§Rotenone & amytal inhibit NADH dehydrogenase in Complex I§Cytochrome oxidase – inhibited by CO, azide (N3

-), cyanide (CN-)

Figure 10.10 Inhibitors of the Electron Transport Chain

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.1: Electron Transport

4H+

4H+

4H+

4H+

Section 10.2: Oxidative Phosphorylation

§Oxidative phosphorylation –energy generated by ETC conserved by phosphorylation of ADP to ATP

§Chemiosmotic coupling theory§Energy released by ETC creates

electrochemical gradient§Gradient drives ATP synthesis

Figure 10.11 Overview of the Chemiosmotic Model

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§Chemiosmotic Theory1. Electrons pass through

ETC§ Protons pumped into

intermembrane space,§ Generates proton motive

force2. Protons move back across

membrane via ATPsynthase driving ATPformation§ Thermodynamic favorable

flow of protonsFigure 10.11 Overview of the Chemiosmotic Model

Section 10.2: Oxidative Phosphorylation

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Evidence for chemiosmotic theory:1. pH drops in a weakly buffered mitochondria

suspension when actively respiring2. Disruption of inner membrane stops respiration3. Uncouplers and ionophores (e.g., gramicidin A)

disrupt the proton gradient, inhibiting ATPsynthesis

Figure 10.12 Uncouplers

Section 10.2: Oxidative Phosphorylation

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

ATP synthase – ‘lollipop’-shaped structure; 2 components

§F1 unit – active ATPase§5 different subunits§3 nucleotide binding catalytic sites§Requires translocation of three

protons §F0 unit -transmembrane channel

§3 different subunits§Inhibited by oligomycin

Figure 10.13 The ATP Synthase

Section 10.2: ATP Synthesis

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§Consists of two rotors linked by a strong flexible stator

§F0 motor converts the protonmotive force into rotational force that drives ATPsynthesis§C ring – revolving component, §e/g subunit – central shaft§Rotates within a,b hexamer of F1

unit§Stator – b/d subunit; prevent

rotation

Figure 10.14 The ATP Synthase From Escherichia coli

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.2: ATP Synthesis

§b subunits of the ATP synthase have three conformations: open (O), tight (T), and loose (L)§Steps:

1. ADP and Pi bind to L site; rotation convertsit to T conformation

2. ATP synthesized3. Rotation converts T site to O site, releasing

ATP

Figure 10.15 ATP Synthesis Model

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.2: ATP Synthesis

§Respiratory control - activates when ADP and Piconcentrations high

§Inhibited when ATP concentrations high

§ADP-ATP translocator -controls amounts of ATP & ADP in mitochondria

§Phosphate carrier (H2PO4-/H+

symporter) – controls amount of H2PO4

-/H+

Figure 10.16 The ADP-ATP Translocator and the Phosphate Translocase

Section 10.2: Control Oxidative Phosphorylation

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§Glycerol phosphate shuttle§cytoplasmic NADH reduces DHAP into glycerol-3-

phosphate§ glycerol-3-phosphate oxides FAD à FADH2§Produces 1.5 ATP

Figure 10.17a Shuttle Mechanisms That Transfer Electrons from Cytoplasmic NADH to the Respiratory Chain

Section 10.2: Oxidative Phosphorylation

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.2: Oxidative Phosphorylation

Figure 10.17b Shuttle Mechanisms That Transfer Electrons from Cytoplasmic NADH to the Respiratory Chain

Malate-aspartate shuttle§ Cytoplasmic NADH

reduces oxaloacetate to malate

§ Transported to matrix§ Malate is reoxidized to

produce NADH§ OAA returned to cytroplasm

via transamination reaction converting it to aspartate

§ Produces 2.25 ATP

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.2: Oxidative Phosphorylation

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§All living processes take place within a redox environment§Redox state is regulated within a narrow range because of redox-

sensitive nature of many pathways§Important linked redox pairs (NAD(P)H/NAD(P)+ and GSH/GSSG)

help maintain redox conditions§GSH (glutathione) is a key cellular-reducing agent

§Reactive oxygen species (ROS)- oxygen accepts single electrons forming unstable derivatives

§Superoxide radical, hydrogen peroxide, hydroxyl radical, singlet oxygen

§Antioxidants interact with ROS to mitigate damage§Under certain conditions, antioxidant mechanisms are

overwhelmed, leading to oxidative stress§Enzyme inactivation, polysaccharide depolymerization, DNA

breakage, membrane destruction§Oxidative damage has been linked to 100 human diseases

Section 10.3: Oxygen , Cell Function, and Oxidative Stress

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§Reactive Oxygen Species§Diatomic oxygen - diradical, meaning it has two unpaired electrons§Electrons can leak out of the ETC and interact with O2

Figure 10.18 Overview of Oxidative Phosphorylation and ROS Formation in the Mitochondrion

Section 10.3: Oxygen , Cell Function, and Oxidative Stress

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§Types of reactive oxygen species:§First created is superoxide

radical (O2●-), which acts as a

nucleophile§O2

●- can react with itself to formhydrogen peroxide H2O2

§H2O2 can react with Fe2+ to formhydroxyl radical (●OH),

which can initiate autocatalytic radical chainreaction

Figure 10.19 Radical Chain Reaction

Section 10.3: Oxygen , Cell Function, and Oxidative Stress

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§H2O2 can react with Fe2+ to form hydroxyl radical (●OH), which can initiate autocatalytic radical chain reaction

§Singlet oxygen (1O2) formedfrom H2O2 or superoxidecan be damaging toaromatics andconjugated alkenes

Section 10.3: Oxygen , Cell Function, and Oxidative Stress

Figure 10.19 Radical Chain ReactionFrom McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§Also reactive nitrogen species (RNS)§Nitric oxide, nitrogen dioxide, and peroxynitrite§Physiological functions of NO include blood pressure

regulation, inhibition of blood clotting, anddestruction of foreign cells by macrophages

Section 10.3: Oxygen , Cell Function, and Oxidative Stress

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§Antioxidant Enzyme Systems§To protect against oxidative stress, living organisms

have developed several antioxidant defensemechanisms

§Four enzymes: superoxide dismutase, glutathioneperoxidase, peroxiredoxin, and catalase§Superoxide dismutase forms H2O2 and O2 from

superoxide radical§Catalase forms H2O and O2 from H2O2

Section 10.3: Oxygen , Cell Function, and Oxidative Stress

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§Glutathione peroxidase uses the reducing agent GSH tocontrol peroxide levels

§Reduces H2O2 to form water and transforms organicperoxides to alcohols

§Glutathione reductase is also an important enzyme in theglutathione system

Figure 10.21 The Glutathione-Centered System

Section 10.3: Oxygen , Cell Function, and Oxidative Stress

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

§Peroxiredoxins (PRX) are a class of enzymes that detoxifyperoxides

§Uses thiol-containing peptides like thioredoxin§Thioredoxin is involved in redox reactions mediated by the

peroxiredoxin/thioreductase system

Figure 10.22 The Thioredoxin-Centered System

Section 10.3: Oxygen , Cell Function, and Oxidative Stress

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press

Section 10.3: Oxygen , Cell Function, and Oxidative Stress

§Antioxidant Molecules§a-Tocopherol (vitamin E) is a potent, lipid-soluble radical scavenger§b-carotene, a carotenoid, is a precursor of vitamin A (retinol): a

potent, lipid-soluble radical scavenger in membranes §Ascorbat(vit C) protects membranes through two mechanisms:

scavenging a variety of ROS in aqueous environments and enhancing the activity of a-tocopherol

Figure 10.23 Selected Antioxidant Molecules

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press