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 energyPhotophosphorylation

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 Phosphorylation19.1 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 -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

Full reduction; 360nm absorption

Partial reduction; 450nm absorption

Full oxidation; 370 & 440 nm absorption

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) electron accept

-High reduction potential (induced by binding

to protein)

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; Ubiquinone

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; Cytochromes

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

Membrane-bound electron carriers; Iron-sulfur proteins

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

Separation of functional complexes of respiratory chain

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

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 3-phosphate & 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

; QN & 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