Electron Transport chain, structure and Organization      طRedox potentials and free energy changes     طCoupling of oxidative phosphorylation to electron transferer      ط

                                       D4 246-261

Electron transfer chain

1. Mitochondrial Structure:fig6.25, fig6.26, Outermembrane, Intramembrane space, innermembrane (F1F0-ATPase), Matrix (actin filaments)

2. In Glycolysis: NADH produced in cytoplasm by GAPDH, a. Reoxidized in cytoplasm to NAD+ by LDH (in RBCs, anaerobic) b. fig 6.28, 7.9, Transported into mitoch by substrate shuttle (aerobic),

* Gly-phopsh: muscle mitoch inner memb, 2 ATP (FADH2)* Mal-Asp: liver mitosol, 3 ATP (NADH)

3. In CAC, FA & KB oxid: NADH & FADH2 produced in cytoplasm by GAPDH,Yields ATP in presence of O2 during electron transport sequence (Respiratory Chain)

Introduction

§ Electron transfer: Electron donor (red) electron donor (oxid):AH2 + B A + BH2

§ In reducing equivalents, both electrons & protons are transferred:NADH + H+ + FAD+ NAD+ + FADH2

§ In cytochromes, only electrons are transferred:cyt c (Fe2+) + cyt a (Fe3+) cyt c (Fe3+) + cyt a (Fe2+)

§ Oxid form & red form are referred as REDOX couple/pair

§ Quantitative elect transf facilitated as oxid-red POTENTIAL, measured as electromotive force (emf)

§ table6.6, the lower the negative emf (–) the more energy produced (ATP) & the higher positive emf (+) the more energy utilized

Oxidation-Reduction Reaction

§ Oxid-red POTENTIAL (with 2 REDOX pair) = Free-Energy Change (in chem. react)(NAD+/NADH : ½O2/H2O) depends on concentration of reactant & product

§ In electectron transfer chain, the Free-Energy from oxid-red POTENTIAL (NADH : O2) generates energy(synthesis of ATP from reducing equivalent) if necessary enzs are present

Free-Energy Change

Mitoch. electectron trasnport (resp chain) major components (oxid-red react):1. NAD+-linked dehydrogenases (NAD+-DHs)2. Flavin-lined dehydrogenases (Flv-DHs)3. Iron-Sulfur Protiens (Fe-S centre)4. Coenzyme Q (CoQ)5. Cytochromes (Cyt)

Multi-component System

In CAC, FA oxid: NAD+ (oxidized form) is reduced to NADH1. Cytoplasm: -  GAPDH -  G6PDH, NAD(P)+: NADP+ is not a substrate in mitoch resp chain but    Used in reductive biosynthesis (FA, Sterols)

2. Mitochondria: -  IsoCDH: Isocitrate + NAD(P)+ α-KG + CO2 + NAD(P)H + H+

-  MDH: L-Malate + NAD+ OA + NADH + H+

§ NAD+ can be measured by spectrophotometer(light absorbance & fluorescence emission)

§ NAD+ enzyme assay for determination of changes in substrate or O2

concentration or during drug or hormone addition(function of metabolic condition)

1. NAD+-linked DHs

fig6.34, Flavin (i.e. FAD+, FMN)derived from riboflavin as elect acceptor (oxid form)

table6.8, Flavin-linked Enzymes:e.g. SDH, DHLDH, NADHDH

§ Flavin (FAD+, FMN) binds tightly noncovalently to their enzyme or protien

§ Flavoproteins:-  Flv-DHs: reduce flavin is reoxidized by electron carriers

(other flavins, CoQ)-  Oxidase: reduced flavin is reoxidized by O2 as electron acceptor and

yields H2O2 (2H2O2 catalase 2H2O + O2)

2. Flavin-linked DHs

§ fig6.35, In flavin-linked enzymes, containing non-heme iron (Fe-S Center), iron is converted from oxid form (Fe3+) to the reduced form (Fe2+) during transform of electron from reducing equivalents

§ SDH & NADHDH contain Fe-S centers found in all species (microorganism & mammals)

3. Fe-S Center

§ The aerobic (O2) energy-generating function possess Cyts involved in elect trasnf system

§ fig6.37, Cyts are prts possess bound iron-containing Heme group

§ unlike hemoglobins or myoglobins Heme Iron (Fe), the Heme Fe of Cyst oxid (Fe3+) or red (Fe2+) acts as a function of electron transport chain

§ fig6.36, Type of Cyts are: a, b & c (c smallest subunit) depends on α band of absorption spectrum and type of Heme

§ fig6.37, since Fe of Heme fill coordination position of Cyt (except Cyt a3), direct O2 binding to Fe can be prevented by inhibitors (cyanide, azide, CO)

5. Cyts

Resp chain is located in inner membrane of mitochfig6.40, fig6.44, Elect trasp is in a specific sequence:§ -  NAD+-linked Enzs (CAC) Reducing Equivalents (electrons) electron Complex I (NADHDH + FMN-linked, Fe-S center), & ejects H+ or-  FAD-lined Enzs (FA, KB oxid) Reducing Equivalents (electrons) electron Complex II (SDH)§ Complex I / II elect CoQ (mobile carrier) elect Complex III (Cyt b, c1, Fe-S center), & ejects H+

§ Complex III elect Cyt c elect Complex IV (Cyt oxidase), & ejects H+

§ ½ O2 is electron acceptor H2O (O2 consumption) proton (H+) ejected from matrix to space are translocated back to matrix by F1F0-ATPase (ATP synthetase process, oxid-phosph):

ADP + Pi ATP

Respiratory Chain

Res

pir

ato

ry C

hai

n

Respiratory Chain

Respiratory Chain

Respiratory Chain

Chemiosmotic theory, ATP synthetase      طInhibitors of respiratory chain     ط Uncouplers: Dinitrophenol, Valinomycin, Thermogenin     ط                                     D4 261-263  ,     L2  555-563

The  respiratory chain and oxidative-phosphorylation

§ fig6.40, The fish poison (retenone) & Barbiturate (amytal) inhibit at NADHDH/flavorprotein

§ The antibiotic (antimycin A) inhibit at Cyt b

§ Cyanide (CN), azide combine with Fe3+ (oxid Heme iron form) at Cyt a and a3, which prevents reduction of Heme iron from Cyt c

§ Carbonmonoxide (CO) binds to Fe2+ (red Heme iron form) at terminal step (Cyt c) of resp chain, which inhibit catalyzation by Cyt oxidase (complex IV)

If electrons are not oxidized causing results in impairment of energy-generation function (ATP synthatase), which leads tissue asphyxia and death

Inhibitors

Reducing equivalents from S-ate can be transferred to NADH with hydrolysis of ATPfig6.46, the electron transport can be reversible:S-ate F-ate (elect donor)  +  FAD FADH2 ……

Reversibility of Electron Transfer

§ fig6.47, Succinate (elect donor) + ADP (phosphate acceptor) ==> active state (rapid)

§ In the active state,-  electrons are transferred (succinate fumarate), elect transp-  O2 is consumped (O2 H2O), oxid-  ATP is synthesized (ADP ATP), phosph

§ This continues till ADP concentration is zero (0) ==> resting state

§ In the resting state,-  all stops: elect transp, O2 is consumption & ATP is synthesis

§ Once ADP is added ==> all continue including ATP synthesis continues till O2 concentration is zero (0)

*  Integrity of mitoch. is required for tight coupling

Coupling of Oxidative Phosphorylation to Electron Transport

§ fig6.48, Succinate + ADP ==> active state (rapid O2 consumption)

§ This continues till Oligomycin (inhibitor of phoph, F1F0-ATPase) is added ==> resting state (all stop because of coupling)

§ When DNP (an uncoupler) is added ==> rapid O2 consumption

§ This continues till O2 concentration is zero (0)

* Other uncouplers: FCCP, Valinomycin, Thermogenin* Uncouplers of resp and phosph act by pumping protons from space back to matrix.  This alters the normal flow of protons through F1F0-ATPase, which lead to no ATP synthesis

Uncouplers

1. Chemical-coupling hypothesis (substrate-level phoph)

2. Conformation-coupling hypothesis 3. Chemiosmotic-coupling Mechanism (storage of energy)

Mechanism of Coupling

1. Chemical-coupling hypothesis (substrate-level phoph):

§ GAPDH (glycolysis):-  GAP oxid to a high-energy intermediate product (1,3BPG)-  ATP is synth in the next step by PGK

§ SCoAS-tase (CAC):     the high-energy product SCoA generates GTP

Mechanism of Coupling

2. Conformation-coupling hypothesis § In muscle contraction

a) ATP hydrolysis drives conformational change in myosin head groupb) This change disrupts cross-bridge to actin thin filaments

§ In elect ransfa) Elect transport through inner membrane causes membrane protein conformational changeb) ATP synthesis during change in membrane protein from high-energy form to low

* given evidence that conformational integrity is involved in the mechanism of ATP synthesis

Mechanism of Coupling

3. Chemiosmotic-coupling Mechanism (storage of energy)

§ fig6.49, Electrochemical gradient (protons) between inner membrane during electron transport

§ This gradient is formed by pumping protons from matrix to space

§ Protons (H+) allowed back to the matrix during ATP synthetaseprocess (F1F0-ATPase)

§ One ATP is synthesized per one proton pumped back from space to matrix

Mechanism of Coupling