* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Biochemistry 6/e
Magnesium in biology wikipedia , lookup
Basal metabolic rate wikipedia , lookup
Magnesium transporter wikipedia , lookup
Western blot wikipedia , lookup
Nicotinamide adenine dinucleotide wikipedia , lookup
Phosphorylation wikipedia , lookup
Mitochondrial replacement therapy wikipedia , lookup
Photosynthesis wikipedia , lookup
Metalloprotein wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Biochemistry wikipedia , lookup
Mitochondrion wikipedia , lookup
Microbial metabolism wikipedia , lookup
Adenosine triphosphate wikipedia , lookup
Photosynthetic reaction centre wikipedia , lookup
Citric acid cycle wikipedia , lookup
Light-dependent reactions wikipedia , lookup
NADH:ubiquinone oxidoreductase (H+-translocating) wikipedia , lookup
Oxidative phosphorylation Structure of mitochondria outer mt. mb.: permeable for molecules Mwt < 5000 g/mol, ‘cause contains porin protein = VDAC = voltage-dependent anion channel inner mt. mb.: is the most impermeable mb. among all mb-s, have biggest protein content, only those ions, molecules can cross that have transporter (except O2, CO, CO2, NO) green = mitoch. Function of mitochondria: the energy factory A sedentery male of 70 kg uses 83 kg ATP/day, but posses only about 250 g ATP. It is possible only if each ATP molecule cycles 300 times /day, meaning ATP is synthetized from ADP + P, then degraded to them. Mitochondria tend to form network mitoch. without ADP during resting respiration mitoch. with ADP during active respiration Mitochondrial DNA contain different amount of genes in different organisms, more and more genes are transfered to nuclear DNA in eucaryotes It is the cause of lous-born typhus. Suggested: all existing mitoch. are derived from ancestor of R.p. as a result of one endosymbiontic event. (circles = DNA) prowazekii Standard redoxelectrode and standard H2-electrode : H2-electrode is anode and Fe2+/Fe3+ electrode is catode ε⁰ = = ε⁰ Direction of reactions in our cells The sequence of components of electron transport chain is determined by their reduction potential NAD + H+ + 2e- → NADH E°’ = - 0.32 V ½ O2 + 2 H+ + 2 e- → H2O E°’ = + 0.82 V ½ O2 + NADH + H+ → H2O + NAD ΔG°’ = - nF Δ E°’ ΔG°’ = - 2×96500×0.82 – [(-2)×96500×(-0.32)] ΔG°’ = - 220 kJ/mol This amount of energy is liberated when 1 atom of oxygen is reduced to water by electrone-transport chain and NADH donates the 2 electrones Synonyms of the names of electron transport chain complexes = complex I = NADH dehydrogenase (trivial name) systemic name (is a wrong name) correct sytemic name is: succinate: coenzyme Q oxidoreductase = complex II = succinate dehydrogenase (trivial n.) systemic name trivial name = complex III = cytochrome c reductase (trivial n.) = complex IV = cytochrome C : O2 oxidoreductase (systemic name) O2 Composition of NADH-CoQ oxidoreductase, = complex I α-ketoglut. isocitrate and other citirc acid cycle dehydrogenases, PDHC, deh. in FA β-oxidation glutamate deh. etc. can produce NADH NADH + Q + 5 H+matrix → NAD+ + QH2 + 4H+cytoplasm FMN and iron-sulfur proteins, the nonheme iron proteins FMN can take up H-atoms in stepwise manner and can take up and donate electron and proton separatly Fe2+ ↔ Fe3+ + e- Ubiquinone, the ubiquitous quinone = coenzyme Q = Q10 Long isoprene tail makes it lipophilic, it is found in the membrane of aerobic bacteria, plants, animals etc. as well. Succinate dehydrogenase, the complex II is the enzyme of citrate cycle comlex III = cytochrome c reductase and Q cycle Fe2+ ↔ Fe3+ + e- Q-cycle makes both electron of ubiquinone to be usful for one electron acceptor of cytochrome c. QH2 + 2 Cyt.cox + 5 H+matrix → Q + 2 Cyt.cred + 4H+cytoplasm Cytochrome b has the same heme as in hemoglobin, cytochrome c1 heme is different, it is attached to Cys. Comlex III = cytochrome c reductase and Q cycle Oxidation of this UQH2 occurs in two steps. First, an electron from UQH2 is transferred to the Rieske protein and then to cytochrome c1. This releases two H+ to the cytosol and leaves UQ ×- , a semiquinone anion form of UQ, at the Qp site. The second electron is then transferred to the bL heme, converting UQ×- to UQ. The Rieske protein and cytochrome c1 are similar in structure; each has a globular domain and is anchored to the inner membrane by a hydrophobic segment. However, the hydrophobic segment is N-terminal in the Rieske protein and C-terminal in cytochrome c1. The electron on the bL heme facing the cytosolic side of the membrane is now passed to the bH heme on the matrix side of the membrane. This electron transfer occurs against a membrane potential of 0.15 V and is driven by the loss of redox potential as the electron moves from bL (Eo' = 0.100V) to bH(Eo' = +0.050V). The electron is then passed from bH to a molecule of UQ at a second quinone-binding site, Qn, converting this UQ to UQ×-. The resulting UQ×- remains firmly bound to the Qn site. This completes the first half of the Q cycle (Figure 21.12a). The second half of the cycle (Figure 21.12b) is similar to the first half, with a second molecule of UQH2 oxidized at the Qp site, one electron being passed to cytochrome c1 and the other transferred to heme bH and then to heme bH. In this latter half of the Q cycle, however, the bH electron is transferred to the semiquinone anion, UQ×- at the Qn site. With the addition of two H+ from the mitochondrial matrix, this produces a molecule of UQH2, which is released from the Qn site and returns to the coenzyme Q pool, completing the Q cycle. The Q Cycle Is an Unbalanced Proton Pump Why has nature chosen this rather convoluted path for electrons in Complex III? First of all, Complex III takes up two protons on the matrix side of the inner membrane and releases four protons on the cytoplasmic side for each pair of electrons that passes through the Q cycle. The apparent imbalance of two protons in for four protons out is offset by proton translocations in Complex IV, the cytochrome oxidase complex. The other significant feature of this mechanism is that it offers a convenient way for a two-electron carrier, UQH2, to interact with the bL and bH hemes, the Rieske protein Fe-S cluster, and cytochrome c1, all of which are oneelectron carriers. Cytochrome oxidase = complex IV 4 Cyt.cred + 8 H+matrix + O2 → 4Cyt.cox + 4H+cytoplasm+ 2H2O 4 2 The conservative structure of cytochrome c is used to draw the evolutionary tree cytochrome c2 photosynthetic bacterium cytochrome c550 Chemiosmotic theory: proton gradient powers ATP synthesis Hypothesis by Peter Mitchell in 1961 Proof by an artificial system intermembrane space inner membrane matrix ATP synthase complex It is a small motor: the rotor continously stirs clockwise: c ring (10-14 c proteins) strictly bound to γε subunit the stator does not move: a + b + δ + 3α + 3β ATP synthase catalyzes ATP formation Enzyme-bound ATP forms readily in the absence of proton-motive force, but ATP is hydrolysed after. Conclusion: proton movement through ATP synthase is necessary for ATP detachment, not for ATP synthesis Paul Boyer: The conformation of β-subunit denpends on which side of γ-subunit is turned to it L = loose conformation entrapes ADP + P T = tight conformation can form ATP and strictly binds it O = open conf. ATP can leave H+ How proton movement stirs the c-ring? H+ When aspartate takes up proton it becomes uncharged and prefers the hydrophobic membrane to interact, therefore the C-ring is stired, the next Asp can take up another H+ then after a whole cycle the H+ leaves the C-ring to the halfchannel of a-subunit Cytoplasmic NADH produced in glycolysis can enter to mitochondria by shuttles Especially prominent in skeletal muscle to provide sufficient energy for contraction Cytoplasmic NADH produced in glycolysis can enter to mitochondria by shuttles glyceraldehyde-3P dehydrogenase in glycolysis malate dehydrogenase ASAT α-ketoglutarate transporter Glu + H+ /Asp antiporter MDH ASAT NADH dehydrogenase = complex I Important in heart and liver ADP-ATP translocase/antiporter ANT= adenine nucleotide transporter ATP used as energy currency in cytoplasm atractyloside bongkrekic acid 15% of mitochondrial inner membrane proteins is this protein ATP produced in matrix side by ATP synthase ~ ¼ of H+ -gradient energy is spent for ATP/ADP exchange Type and function of substrate anion carriers, ion transporters energy requiring processes lipid synthesis gluconeogenesis And other amino acid, nucleotide, coenzyme, fatty acid transporters ~ 40 types all together PDHC ATP synthesis H2PO4- imb. →HPO42-matrix + H+ Transports protons into mito. And not OH- is exchanged. Biochimica et Biophysica Acta (BBA) – Bioenergetics Volume 1777, Issues 7–8, July–August 2008, Pages 564–578 Or 5, if malate-Asp shuttle works Or 32 What Is the P/O Ratio for Mitochondrial Electron Transport and Oxidative Phosphorylation? The P/O ratio is the number of molecules of ATP formed in oxidative phosphorylation (No of P built into ATP) per two electrons flowing through a defined segment of the electron transport chain (the number of oxygen atom that is reduced to water by respiratory chain: 4 e- + 4 H+ + O2 = 2 H2O). If we accept the value of 10 H+ transported out of the matrix (4 + 2 + 4 H+ by 1. 3. 4. complex) per 2 e- passed from NADH to O2 through the electron transport chain, and also agree (as above) that 4 H+ are transported into the matrix per ATP synthesized and translocated (1/4 of proton gradient is for adenine nucleotide exchange), then the mitochondrial P/O ratio is 10/4 = 2.5, for the case of electrons entering the electron transport chain as NADH. This is somewhat lower than earlier estimates, which placed the P/O ratio at 3 for mitochondrial oxidation of NADH. For the portion of the chain from succinate/glycerol-3P/fatty acyl-CoA dehydr. to O2, the H+/2 e- ratio is 6 (the 1st complex does not work), and the P/O ratio if FADH2 is oxidized would be 6/4 = 1.5; earlier estimates placed this number at 2. The consensus of experimental measurements of P/O ratios for these two cases has been closer to the more modern values of 2.5 and 1.5. Many biochemists have been reluctant to accept the notion of nonintegral P/O ratios. At some point, as we learn more about these complex coupled processes, it may be necessary to reassess the numbers. Calculation of the energy of electrochemical proton gradient ΔG = RT ln c2/c1 + zFΔE ΔG = 8.3×10-3 kJ/molK× 310 K ×2.3 ×1.4 + 1 × 96500 kJ/molV × 0.14 V ΔG = 21.8 kJ/mol energy is necessary to transport out 1 H+ from matrix to intermembrane space (at body temperature, when pH difference is the usual 1.4 and membrane potential is 0.14 V) Hypothetically speaking, how much energy does a eukaryotic cell extract from the glucose molecule? Taking a value of 50 kJ/mol for the hydrolysis of ATP under cellular conditions, the production of 32 ATP per glucose oxidized yields 1600 kJ/mol of glucose . The cellular oxidation (combustion) of glucose yields ΔG = -2937 kJ/mol. We can calculate an efficiency for the pathways of complete oxidation of glucose by glycolysis, PDHC, the TCA cycle, electron transport, and oxidative phosphorylation of Regulation of oxidative phosphorylation Electron-transport chain and ATP synthesis are tightly coupled, the ETC is a near equilibrium process: the supply of reducing equivalents/substrates, of ADP and P determine the speed of electon transport and ATP synthesis as well. Uncopuling proteins generate heat, uncoupler molecules are poisons cold →CNS → adrenalin proton gradient is dissipated without ATP synthesis, only heat is generated by: UCP-1 = thermogenin in brown adipocyte mitochondrial inner mb. UCP-2: many tissues UCP-3: muscle, brown fat Uncoupling molecule: can transport protons back to mitochondria, ETC is accelerated, but no ATP synthesis, just heat Inhibitors of respiratory chain, ATP synthase and transporters atractyloside bongkrekic acid oligomycin DCCD The inhibitory actions of cyanide and azide at this site are very potent, whereas the principal toxicity of carbon monoxide arises from its affinity for the iron of hemoglobin. Herein lies an important distinction between the poisonous effects of cyanide and carbon monoxide. Because animals (including humans) carry many, many hemoglobin molecules, they must inhale a large quantity of carbon monoxide to die from it. These same organisms, however, possess comparatively few molecules of cytochrome a3. Consequently, a limited exposure to cyanide can be lethal. The sudden action of cyanide attests to the organism's constant and immediate need for the energy supplied by electron transport. Oligomycin and DCCD Are ATP Synthase Inhibitors Inhibitors of ATP synthase include dicyclohexylcarbodiimide (DCCD) and oligomycin (Figure 21.29). DCCD bonds covalently to carboxyl groups in hydrophobic domains of proteins in general, and to a glutamic acid residue of the c subunit of Fo×, the proteolipid forming the proton channel of the ATP synthase, in particular. If the c subunit is labeled with DCCD, proton flow through Fo× is blocked and ATP synthase activity is inhibited. Likewise, oligomycin acts directly on the ATP synthase. By binding to a subunit of Fo×, oligomycin also blocks the movement of protons through Fo×. Rotenone is a common insecticide that strongly inhibits the NADH-UQ reductase. Rotenone is obtained from the roots of several species of plants. Tribes in certain parts of the world have made a practice of beating the roots of trees along riverbanks to release rotenone into the water, where it paralyzes fish and makes them easy prey. Ptericidin, Amytal, and other barbiturates, mercurial agents, and the widely prescribed painkiller Demerol also exert inhibitory actions on this enzyme complex. All these substances appear to inhibit reduction of coenzyme Q and the oxidation of the Fe-S clusters of NADH-UQ reductase. 2-Thenoyltrifluoroacetone and carboxin and its derivatives specifically block Complex II, the succinate-UQ reductase. Antimycin, an antibiotic produced by Streptomyces griseus, inhibits the UQ-cytochrome c reductase by blocking electron transfer between bH and coenzyme Q in the Qn site. Myxothiazol inhibits the same complex by acting at the Qp site. Cyanide, Azide, and Carbon Monoxide Inhibit Complex IV Complex IV, the cytochrome c oxidase, is specifically inhibited by cyanide (CN-), azide (N3-), and carbon monoxide (CO). Cyanide and azide bind tightly to the ferric form of cytochrome a3, whereas carbon monoxide binds only to the ferrous form. SUPPLEMENT, not obligatory material SUPPLEMENT, not obligatory material Mitochondria, ER, peroxisome, cytoplasm generates reactive oxigen species (ROS) Mitochondrial ROS production and defense. SUPPLEMENT, not obligatory material Figure 2. Superoxide (O2.-) generated by a respiratory chain is mostly released to a matrix at complex I and IMS at complex III (indicated by stars). O2.- can naturally dismute to hydrogen peroxide (H2O2) or is enzymatically dismuted by matrix MnSOD (1) or Cu/ZnSOD (2) in a IMS or cytosol. H2O2 is detoxified in a matrix by catalase (3), a thioredoxin/thioredoxin peroxidase system (4), or a glutathione/glutathione peroxidase system (5). Alternately, H2O2 can react with metal ions to generate a highly reactive hydroxyl radical (.OH) via Fenton chemistry (6). O2.- is not membrane permeable but can pass through ion channels (solid lines), whereas H2O2 can pass freely through membranes (dashed lines). IMM, inner mitochondrial membrane; IMS, intermembrane space; OMM, outer mitochondrial membrane; O2.-, superoxide; H2O2, hydrogen peroxide; MnSOD, manganese superoxide dismutase; Cu/ZnSOD, copper/zinc superoxide dismutase; CAT, catalase; THD, NADH transhydrogenase; TR, thioredoxin reductase; TPx, thioredoxin peroxidase; TRxred, reduced thioredoxin; TRxox, oxidized thioredoxin; GSH, glutathione; GSSG, glutathione disulfide; IMAC, inner membrane ion channel; VDAC, voltage dependant anion channel; ∆Ψm, membrane potential [Frontiers in Bioscience 14, 1182-1196, January 1, 2009] SUPPLEMENT, not obligatory material [Frontiers in Bioscience 14, 1182-1196, January 1, 2009] Figure 1. Mitochondrial Ca2+ dynamics and stimulation of the TCA cycle and oxidative phosphorylation. Metabolite and ion transport are represented by thin arrows passing through membrane carriers, metabolic pathways by thick arrows, respiratory chain electron transport by broken arrows, and TCA cycle enzymes by shaded ovals. Mitochondrial Ca2+ influx and efflux mechanisms are displayed in yellow. Enzymes and complexes stimulated by Ca2+ are displayed in green, and the respiratory chain is displayed in blue. The mitochondrial outer membrane has been omitted for clarity. MCU, mitochondrial calcium uniporter; mRrR, mitochondrial ryanodine receptor; RAM, rapid mode; PTP, permeability transition pore; ANT, adenine nucleotide transporter; PDH, pyruvate dehydrogenase; CS, citrate synthase; ACON, aconitase; ICDH, isocitrate dehydrogenase; a-KGDH,a-ketogluterate dehydrogenase; SDH, succinate dehydrogenase; FUM, fumarase; MDH, malate dehydrogenase; DYm, membrane potential