Aerobic Metabolism II: Electron Transport Chapter 10 and Oxidative Phosphorylation Overview n Electron Transport – electron transfer to oxygen n Oxidative Phosphorylation – conversion of ADP to ATP n Chemiosmotic coupling – drives synthesis of ATP

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.1: Electron Transport

Series 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

Figure 10.1 The Electron §Chemiosmotic coupling – results Transport Chain from stored potential energy, basis for coupling between oxidation & phosphorylation 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 from

NADH to ubiquinone (CoQ) CoQH2 §NADH dehydrogenase complex §Large complex - >20 subunits §1 molecule FMN, 7 Fe clusters §Flavin mononucleotide (FMN) oxides

NADH à FMNH2

+ + NADH + H E-FMN àNAD + E-FMNH2 à

Feox à Fered à CoQ à CoQH2

Figure 10.2 Two Iron-Sulfur Clusters

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

Figure 10.4 Electron Movement through Complex I of the Electron Transport Chain

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

Figure 10.7 Electron Transport through Complex III

- 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 matrix +3 + +2 + CoQH2 = 2cyt cox (Fe ) = 2H matrix à CoQ + 2cyt cred(Fe ) + 4H cytosol

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.1: Electron Transport

- 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

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

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

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.1: Electron Transport Section 10.1: Electron Transport

Figure 10.9 Energy Relationships in the Electron Transport Chain

§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

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.1: Electron Transport Inhibitors

Figure 10.10 Inhibitors of the Electron Transport Chain

§When electron transport is inhibited, O2 consumption is reduced 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 )

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 Section 10.2: Oxidative Phosphorylation

§Chemiosmotic Theory 1. Electrons pass through ETC § Protons pumped into intermembrane space, § Generates proton motive force 2. Protons move back across membrane via ATP synthase driving ATP formation § Thermodynamic favorable flow of protons Figure 10.11 Overview of the Chemiosmotic Model From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.2: Oxidative Phosphorylation

Figure 10.12 Uncouplers

Evidence for chemiosmotic theory: 1. pH drops in a weakly buffered mitochondria suspension when actively respiring 2. Disruption of inner membrane stops respiration 3. Uncouplers and ionophores (e.g., gramicidin A) disrupt the proton gradient, inhibiting ATP synthesis

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.2: ATP Synthesis

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

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.2: ATP Synthesis

§Consists of two rotors linked by a strong flexible stator

§F0 motor converts the proton motive force into rotational force that drives ATP synthesis §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 converts it to T conformation 2. ATP synthesized 3. 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: Control Oxidative Phosphorylation

§Respiratory control - activates

when ADP and Pi concentrations 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

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.2: Oxidative Phosphorylation

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

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

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

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.2: Oxidative Phosphorylation

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

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

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 Section 10.3: Oxygen , Cell Function, and Oxidative Stress §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 From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.3: Oxygen , Cell Function, and Oxidative Stress

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

§Reactive Oxygen Species §Diatomic oxygen - diradical, meaning it has two unpaired electrons

§Electrons can leak out of the ETC and interact with O2

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.3: Oxygen , Cell Function, and Oxidative Stress

§Types of reactive oxygen species: §First created is superoxide ●- radical (O2 ), which acts as a nucleophile ●- §O2 can react with itself to form hydrogen peroxide H2O2 2+ §H2O2 can react with Fe to form hydroxyl radical (●OH), which can initiate autocatalytic radical chain reaction

Figure 10.19 Radical Chain Reaction From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.3: Oxygen , Cell Function, and Oxidative Stress

2+ §H2O2 can react with Fe to form hydroxyl radical (●OH), which can initiate autocatalytic radical chain reaction 1 §Singlet oxygen ( O2) formed from H2O2 or superoxide can be damaging to aromatics and conjugated alkenes

Figure 10.19 Radical Chain Reaction From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.3: Oxygen , Cell Function, and Oxidative Stress

§Also reactive nitrogen species (RNS) §Nitric oxide, nitrogen dioxide, and peroxynitrite §Physiological functions of NO include blood pressure regulation, inhibition of blood clotting, and destruction of foreign cells by macrophages

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.3: Oxygen , Cell Function, and Oxidative Stress

§Antioxidant Enzyme Systems §To protect against oxidative stress, living organisms have developed several antioxidant defense mechanisms §Four enzymes: superoxide dismutase, glutathione peroxidase, peroxiredoxin, and catalase

§Superoxide dismutase forms H2O2 and O2 from superoxide radical

§Catalase forms H2O and O2 from H2O2

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.3: Oxygen , Cell Function, and Oxidative Stress Figure 10.21 The Glutathione-Centered System

§Glutathione peroxidase uses the reducing agent GSH to control peroxide levels

§Reduces H2O2 to form water and transforms organic peroxides to alcohols §Glutathione reductase is also an important enzyme in the glutathione system

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.3: Oxygen , Cell Function, and Oxidative Stress Figure 10.22 The Thioredoxin-Centered System

§Peroxiredoxins (PRX) are a class of enzymes that detoxify peroxides §Uses thiol-containing peptides like thioredoxin §Thioredoxin is involved in redox reactions mediated by the peroxiredoxin/thioreductase system

From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press Section 10.3: Oxygen , Cell Function, and Oxidative Stress

Figure 10.23 Selected Antioxidant Molecules

§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

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