Chapter 19 Oxidative Phosphorylation and Photophosphorylation

Oxidative Phosphorylation

 In mitochondria 

Reduction

of O 2 NADH or FADH 2 to H 2 O with electrons from  Independent on the light energy

Photophosphorylation

 In chloroplast 

Oxidation

of H 2 O to O 2 acceptor  with NADP + Dependent on the light energy as electron

Oxidative Phosphorylation vs. Photophosphorylation



Similarities

 Flow of electrons through a chain of membrane-bound carriers

(Downhill: exogernic process)

 Proton transport across a proton-impermeable membrane

(Uphill: endogernic process)

Free energy from electron flow is coupled to generation of proton gradient across membrane 

Transmembrane electrochemical potential (conserving free energy of fuel oxidation)

 “

Chemiosmotic theory

by Peter Mitchell (1961)” Proton gradient as a reservoir of energy generated by biological oxidation  ATP synthase couples proton flow to ATP synthesis

Oxidative Phosphorylation

19.1 Electron-Transfer Reactions in Mitochondria

Mitochondria

 

Site of oxidative phosphorylation

 Eugene Kennedy and Albert Lehninger (1948)

Structure

 Outer membrane  Free diffusion of small molecules (Mr < 5,000) and ions through porin channels  Inner membrane  Impermeable to most small molecules and ions (protons)   Selective transport Components of the respiratory chain and the ATP synthase  Mitochondria matrix  Contain enzymes for metabolism  Pyruvate dehydrogenase complex    Citric acid cycle b -oxidation Amino acid oxidation

Electron transfer in biological system



Types of electron transfer in biological system

 Direct electron transfer : Fe 3+  Fe 2+    Hydrogen atom (H + Hydride ion (:H ) Organic reductants + e )

* Reducing equivalent

 A single electron equivalent transferred in an redox reaction 

Types of electron carriers

 NAD(P) +     FAD or FMN Ubiquinone (coenzyme Q , Q) Cytochrome Iron-sulfur proteins

NAD(P) + & FAD/FMN ; universal electron acceptors NAD(P) +

-Cofactors of dehydrogenases (generally) -Electron transfer as a form of :H -Low [NADH]/[NAD + ]  catabolic reactions -High [NADPH]/[NADP + ]  anabolic reactions -No transfer into mito matrix -Shuttle systems (inner mito membrane) Partial reduction; 450nm absorption Full oxidation; 370 & 440 nm absorption

FAD/FMN (flavin nucleotides)

-Tightly bound in flavoprotein (generally) -One (semiquinone) or two (FADH 2 or FMNH 2 ) Full reduction; 360nm absorption electron accept -High reduction potential (induced by binding to protein)

Membrane-bound electron carriers ; Ubiquinone

    

Coenzyme Q or Q Lipid-soluble benzoquinone with long isoprenoid side chain Accept one (semiquinone radical; • QH) or two electrons (ubiquinol; QH 2 ) Freely diffusible within inner mito membrane

 Shuttling reducing equivalents between less mobile electron carriers

Coupling electron flow to proton movement

Membrane-bound electron carriers ; Cytochromes

   Iron-containing heme prosthetic group 3 classes of Cyt in mitochondria (depending on differences in light-absorption spectra) ;

a (near 600nm), b (near 560nm), c (near 550nm)

Cyt

c

- Covalently-attached heme through Cys - Soluble protein associated with outer surface of inner mito membrane

Membrane-bound electron carriers ; Iron-sulfur proteins

   Irons associated with inorganic S or S of Cys One electron transfer by redox reaction of one iron atom > 8 Fe-S proteins involved in mito electron transfer  Reduction potential of the protein : -0.65 V ~ +0.45 V

Determining the Sequence of Electron Transfer Chain



Based on the order of standard reduction potential (

E’

°

)

  Electron flow from lower NADH  Q  Cyt

b



E’

° Cyt

c 1

to higher

E’

 Cyt

c

 ° Cyt

a

 Cyt

a 3

 O 2

Determining the Sequence of Electron Transfer Chain

 

Reduction of the entire chain of carriers



sudden addition of O 2

  Spectroscopic measurement of oxidation of each electron carriers Closer to O 2  faster oxidation

Inhibitors

 Blocking the flow of electrons  Before/after the inhibited step : fully reducted/ fully oxdized

Electron Carriers in multienzyme complex



Membrane-embedded supramolecular complexes (organized in mito respiratory chain)

 Complex I : NADH  Q    Complex II : Succinate  Complex III : Q  Complex IV : Cyt Cyt

c

 to O 2 Q 

Separation of functional complexes of respiratory chain

Electron Carriers in multienzyme complex

Path of electrons from various donors to ubiquinone

Complex I : NADH:ubiquinone oxidoreductase (NADH dehydrogenase)

 

42 polypeptide chains

 FMN-containing flavoprotein  > 6 iron sulfur centers

Functions : proton pump driven by the energy from electron transfer

 Exergonic transfer of :H from NADH and a proton from the matrix to Q  NADH + H + + Q  NAD + + QH 2  Endergonic transfer 4 H + from the matrix to the intermembrane space  NADH + 5H N + + Q  NAD + + QH 2 + 4H p + 

Inhibitors : e -



flow from Fe-S center

Amytal (a barbiturate drug)   Rotenone (plant, insecticide) Piericidin A (antibiotic)

Complex II : Succinate Dehydrogenase

 

Only membrane-bound enzyme in the citric acid cycle Structure

  4 subunits C and D : transmembrane side  Heme

b

: preventing electron leakage to form reactive oxygen species   Q binding site  A and B : matrix side  Three 2Fe-2S centers   FAD Binding site of succinate Electron passage : entirely 40 Å long (< 11 Å of each step)

Electron transfer from Glycerol 3 phosphate & fatty acyl-CoA

  Electron from fatty acyl-CoA  FAD  (ETF)  electron-transferring flavoprotein ETF: ubiquinone oxidoreductase  Q Electron from glycerol 3-phosphate  FAD in glycerol 3-phosphate dehydrogenase  Q

 Shuttling reducing equivalents from cytosolic NADH into mito matrix ; glycerol 3-phosphate dehydrogenase

Complex III: Cyt bc

1

complex (Q:Cyt c oxidoreductase)

  

e H + transfer (ubiquinol (QH 2 )



transfer (matrix



Cyt c) intermembrane space) Dimer of identical monomers (each with 11 different subunits) Functional core of each monomer; cyt b (2 heme; b

H

iron-sulfur protein (2Fe-2S center) + cyt c

1

(heme c

1

) & b

L

) + Rieske

Complex III: Cyt bc

1

complex (Q:Cyt c oxidoreductase)

 

Two binding sites for ubiquinone ; Q N & Q P Antimycin A

: binding at Q N 

Myothiazol

: binding at Q P  block e block e flow (heme b H  flow (QH 2  Q) Rieske iron-sulfur protein)

Cavern (space at the interface between monomers) ; Q N & Q P are located

Q cycle in complex III



Two stages 1 st 2 nd stage; Q (on N side)



semiquinone radical stage; semiquinone radical



QH 2

Complex IV : Cytochrome Oxidase

 

e transfer from cyt c to O 2



H 2 O Structure;

13 subunits  Subunit II; 2 Cu ions complexed with –SH of 2 Cys (Cu A )   Subunit I; 2 heme groups,

a

&

a 3

Cu ion (Cu B ) 

a 3

+ Cu B  2 nd 1 st binuclear center binuclear center

Complex IV : Cytochrome Oxidase



Electron transfer

  Cyt

c

4 Cyt 

c

Cu A  heme (red) + 8 H N +

a

 heme

a 3

-Cu B + O 2  4 cyt

c

center (ox) + 4H p +  O + 2 H 2 2 O  4H N + as substrate, 4H N + for pumping out