Chapter 19
Oxidative Phosphorylation and
Photophosphorylation
Oxidative Phosphorylation
 In mitochondria
 Reduction of O2 to H2O with electrons from
NADH or FADH2
 Independent on the light energy
Photophosphorylation
 In chloroplast
 Oxidation of H2O to O2 with NADP+ as electron
acceptor
 Dependent on the light energy
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 : Fe3+  Fe2+
Hydrogen atom (H+ + e-)
Hydride ion (:H-)
Organic reductants
* 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)
FAD/FMN (flavin nucleotides)
-Tightly bound in flavoprotein (generally)
-One (semiquinone) or two (FADH2 or FMNH2)
Partial reduction;
450nm
absorption
Full reduction;
360nm
absorption
electron accept
-High reduction potential (induced by binding
to protein)
Full oxidation;
370 & 440 nm
absorption
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; QH2)
 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 E’° to higher E’°
 NADH  Q Cyt b  Cyt c1  Cyt c  Cyt a  Cyt a3  O2
Determining the Sequence of Electron
Transfer Chain
 Reduction of the entire chain of carriers
 sudden addition of O2
 Spectroscopic measurement of oxidation of each electron carriers
 Closer to O2  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  Q
Complex III : Q  Cyt c
Complex IV : Cyt  to O2
 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+ + QH2
Endergonic transfer 4 H+ from the matrix to
the intermembrane space
 NADH
+ 5HN+ + Q  NAD+ + QH2 +
4Hp+
 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 3phosphate & fatty acyl-CoA


Electron from fatty acyl-CoA
 FAD  electron-transferring flavoprotein
(ETF)  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 bc1 complex
(Q:Cyt c oxidoreductase)
 e- transfer (ubiquinol (QH2)  Cyt c)
H+ transfer (matrix  intermembrane space)
 Dimer of identical monomers (each with 11 different subunits)
 Functional core of each monomer; cyt b (2 heme; bH & bL) + Rieske
iron-sulfur protein (2Fe-2S center) + cyt c1 (heme c1)
Complex III: Cyt bc1 complex
(Q:Cyt c oxidoreductase)
 Two binding sites for ubiquinone
; Q N & QP
Antimycin A: binding at QN block e- flow (heme bH Q)
Myothiazol: binding at QP block e- flow (QH2  Rieske iron-sulfur protein)
 Cavern (space at the interface between monomers)
; QN & QP are located
Q cycle in complex III
 Two stages
1st stage; Q (on N side)  semiquinone radical
2nd stage; semiquinone radical  QH2
Complex IV : Cytochrome Oxidase
 e- transfer from cyt c to O2  H2O
 Structure; 13 subunits
 Subunit II; 2 Cu ions complexed with –SH of 2 Cys (CuA)  1st binuclear center
 Subunit I; 2 heme groups, a & a3
Cu ion (CuB)
 a3 + CuB  2nd binuclear center
Complex IV : Cytochrome Oxidase
 Electron transfer


Cyt c  CuA  heme a  heme a3-CuB center  O2
4 Cyt c (red) + 8 HN+ + O2  4 cyt c (ox) + 4Hp+ + 2 H2O
 4HN+ as substrate, 4HN+ for pumping out