Chapter 19 Oxidative Phosphorylation and Photophosphorylation

Oxidative Phosphorylation  In mitochondria  Reduction of O 2 to H 2 O with electrons from NADH or FADH 2  Independent on the light energy Photophosphorylation  In chloroplast  Oxidation of H 2 O to O 2 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   -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 + + 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 (FADH 2 or FMNH 2 ) 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; 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 ; 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 : V ~ 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 c 1  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 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 O 2

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  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 bc 1 complex (Q:Cyt c oxidoreductase) e - transfer (ubiquinol (QH 2 )  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; b H & b L ) + Rieske iron-sulfur protein (2Fe-2S center) + cyt c 1 (heme c 1 )

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  block e - flow (heme b H  Q) Myothiazol: binding at Q P  block e - flow (QH 2  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 stage; Q (on N side)  semiquinone radical 2 nd 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 )  1 st binuclear center  Subunit I; 2 heme groups, a & a 3 Cu ion (Cu B )  a 3 + Cu B  2 nd binuclear center

Complex IV : Cytochrome Oxidase Electron transfer  Cyt c  Cu A  heme a  heme a 3 -Cu B center  O 2  4 Cyt c (red) + 8 H N + + O 2  4 cyt c (ox) + 4H p H 2 O  4H N + as substrate, 4H N + for pumping out