3 The mitochondrial ultrastructure Palade, G. (1952) The fine structure of mitochondria. Anat. Rec. 114, 427-451 Sjöstrand, F.S. (1956) The ultrastructure of cells as revealed by the electron microscope. Int. Rev. Cytol. 5, 455-533
5 Fig. 2. Tomography of an isolated rat-liver mitochondrion. (a) Surface-rendered 3D image of an isolated rat-liver mitochondrion. C, cristae; IM, inner boundary membrane; OM, outer membrane. Arrowheads point to tubular regions of cristae that connect them to IM and to each other. (b) Region of a 5-nm slice from the same tomogram showing numerous contact sites between OM and IM. Arrow points to particle bridging OM with attached vesicle of putative endoplasmic reticulum. A VRML version of the 3D model can be found at http://www.wadsworth.org/spider_doc/spider/vrml/uncomp/mito/mito.wrl. Bar, 0.4 µ m. http://www.wadsworth.org/spider_doc/spider/vrml/uncomp/mito/mito.wrl
6 Fig. 3. Computer models generated from segmented 3D tomograms of a mitochondrion in chick cerebellum. (a) The entire model showing all cristae in yellow, the inner boundary membrane in light blue, and the outer membrane in dark blue. (b) Outer membrane, inner boundary membrane and four representative cristae in different colors. Quicktime videos of these models can be found at http://www.sci.sdsu.edu/tfrey/mitomovie.htmhttp://www.sci.sdsu.edu/tfrey/mitomovie.htm
7 Fig. 4. A single section through the 3D tomogram of the mitochondrion used to create the model in Fig. 3. Examples of the various structural features are outlined, identified and enlarged to the left and right, along with their dimensions averaged from the complete tomograms of multiple mitochondria reconstructed in situ in neural tissue, brown adipose tissue and Neurospora crassa.Fig. 3
9 1948 Eugene Kenedy & Albert Lehninger → mitochondria is the site of oxidative phosphorylation. OM Two membranes: OM permeable up to M r <5,000 IM IM impermeable; only by specific transporters First step of oxidative phosphorylation → entry of electron into the respiratory chain Electron source: nicotinamide nucleotides (NAD + or NADP + ) flavin nucleotides (FMN or FAD)
10 NAD/NADP-linked dehydrogenases NAD/NADP-linked dehydrogenases: Reduced subst. + NAD + ↔ oxidized subst. + NADH + H + Reduced subst. + NADP + ↔ oxidized subst. + NADPH + H + Reversible association with proteins NADH → anabolic reactions NADPH → catabolic reactionsFlavoproteins: Covalently bound flavin Stand. reduction potential → depends on the protein
12 Mitochondrial resp. chain → series of sequentially acting e - carries Proteins with prost. groups accepting and donating e - Types of e - transfer: (1) direct transfer; Fe 3+ to Fe 2+ (2) transfer as hydrogen (H + + e - ) (3) transfer as hydride ion (:H - ) Electron carriers in the respiratory chain: (1) Ubiquinone (2) cytochromes (3) Fe-S proteins
13 UQ → coenzyme Q Small; hydrophobic → freely diffusible within the lipid bilayer
15 Cytochromes Cytochromes → characteristic absorption due to heme groups Three types → a, b, c Heme of a and b → tightly but not covalently bound Heme of c → covalently bound through Cys residues. Cytochromes type a, b and some c → intergal proteins of inner membrane Cytochrome c → soluble protein associates to the outer surface of the inner mebrane
19 Direction of e - flow The sequence of e - carriers is determined by their reduction potential Electrons flow spontaneously from carriers with lower E’ to carriers with higher E’ . Determining the standard reduction potential experimentally The order of carriers: NADH → Q → cytochrome b → cytochrome c 1 → cytochrome c → cytochrome a → cytochrome a 3 → O 2
26 Complex I. NADH:ubiquinone oxidoreductase or NADH dehydrogenase 43 polypeptide; FMN-containing flavoprotein plus 6 Fe-S center Catalyses two reactions: (1)NADH + + H + + Q NAD + + QH 2 (2)4H + N 4H + P Second reaction is vectorial moves protons from one location (matrix) to an other (IMS) Overall reaction: NADH + + 5H + N + Q NAD + + QH 2 + 4H + P Ubiquitinol (QH 2 ) diffuses in the inner membrane to Complex III Inhibitors: amytal (barbiturate), rotenone, piericidin A
28 Complex II. Succinate dehydrogenase Only membrane bound enzyme in citric acid cycle 5 prosthetic group; 4 prot. subunits Heme b not part of e - transfer; it reduces e - leakage and ROS production
31 cytochrome bc 1 complex or ubiquinone:cytochrome c oxido- reductase dimer, each containing 11 subunits functional core: cytochrome b (heme b L and b H ), Rieske Fe-S protein (two Fe-S centers), cytochrome c 1 (heme) transfers e - from QH 2 to cytochrome c with vectorial transport of protons from matrix to IMS Net equation for Q cycle: QH 2 + 2 cyt c 1 (oxidised) + 2H + N Q + 2 cyt c 1 (reduced) + 4H + P Inhibitors: Antymicin A (Q N site), Myxothiazol (Q P site) Complex III.
34 Complex IV. Complex IV: cytochrome oxidase Carries e - from cyt c to O 2 reducing it to H 2 O 13 subunits 3 are critical Subunit I two heme groups (a and a3) and a Cu ion (Cu B ) Subunit II two Cu ions complexed with –SH of Cys in a binuclear center (Cu A ) Subunit III essential; role not clear Electron transfer: Cyt c Cu A center heme a heme a 3 -Cu B center O 2 Overall reaction of Complex IV: 4 cyt c (red.) + 8H + N + O 2 4 cyt c (ox.) + 4 H + P + 2H 2 O
41 Energy of e - transfer is conserved in a H + gradient NADH + H + + ½ O 2 NAD + + H 2 O standard free energy change G’ = -220 kJ/mol for NADH (-150 kJ/mol for succinate) Most of this energy is used to pump out protons from the matrix Vectorial Equation: NADH + 11 H + N + ½ O 2 NAD + + 10 H + P + H 2 O Energy stored proton motive force; has two components (1)chemical potential energy difference in H + conc. ( pH = 0.75) (2)electrical potential energy separation of charge ( = 0.15 – 0.20 V)
42 The mitochondrial energy production II. The ATP synthesis Gabor Varbiro
43 Electron transfer releases energy conserved by the proton motive force 200 kJ/mol energy stored from 1 mol of NADH oxidation 50 kJ/mol energy required for the formation of 1 mol of ATP What chemical mechanism couples proton flux to phosphorylation? THE CHEMIOSMOTIC MODEL 1961
50 Mitochondrial ATP synthase F-type ATP-ase catalyzes ADP + P i ATP; accompanied by flow of H+ from P to N side (IMS matrix) Two components: (1) F 1 peripheral membrane protein (2) F o (oligomycin sensitive) integral to membrane When in vesicles F 1 is removed NADH to O 2 e - transfer, but no proton gradient F o has a proton pore Isolated F 1 catalyzes ATP hydrolysis When purified F 1 is added to vesicles reassociates with F o restores ATP synthesis
51 F 1 solubilized from mitotochondria is incubated with ATP in the presence of 18 O-labeled water After a few minutes P i contains 3-4 18 O atoms both ATP hydrolysis and synthesis have occurred several times. ADP + P i ATP is in equilibrium when substrates are bound to ATP synthase Hydrolysis of ATP in free solution results in G’ = -30.5 kJ/mol The difference binding energy of ATP-synthase drives the equlibrium towards ATP production.
52 Proton gradient causes the enzyme to release the ATP formed on its surface.
53 F 1 9 subunits: 3 3 Knoblike shape 8 nm high 10 nm diameter alternating and subunits one domain of subunit making central shaft, passing through F 1 ; other subunit associating with -empty the conformation of the 3 subunits differ due to the association with subunit structure of and subunits not yet revealed
57 F o complex Forming the proton pore: three subunits a, b and c Proportion: ab 2 c 10-12 Subunit c: small (M r 8,000) hydrophobic; two transmembrane helices a small loop at the matrix side Two concentric circles formed by c subunits the and subunits of F1 form a leg-and-foot and stands on the ring of c subunits