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MITOCÔNDRIAS Edson Rosa Pimentel Mitocôndrias – túbulo renal – Coloração de Régaud A B C A: suprarenal B: epidídimo C: Célula adiposa de morcego hibernando A B C Mitocôndrias em espermatozóide (A), e em célula muscular (B,C). Mitochondrial fission and fusion. These processes involve both outer and inner mitochondrial membranes. (A) During fusion and fission, both matrix and intermembrane space compartments are maintained. Different membrane fusion machines are thought to operate at the outer and inner membranes. Conceptually, the fission process resembles that of bacterial cell division. The pathway shown has been postulated from static views such as that shown in (B). (B) An electron micrograph of a dividing mitochondrion in a liver cell. (B, courtesy of Daniel S. Friend.) Dynamic mitochondrial reticulum. (A) In yeast cells, mitochondria form a continuous reticulum underlying the plasma membrane. (B) A balance between fission and fusion determines the arrangement of the mitochondria in different cells. (C) Time-lapse fluorescent microscopy shows the dynamic behavior of the mitochondrial network in a yeast cell. In addition to shape changes, the network is constantly remodeled by fission and fusion (red arrows). The pictures were taken at 3-minute intervals. (A and C, from J. Nunnari et al., Mol. Biol. Cell 8:1233 1242, 1997, with permission by the American Society for Cell Biology.) Interação entre mitocôndria e microtúbulos em célula humana em cultura. A: célula tratada com rodamina mostrando mitocôndria. B: mesma célula com imunofluorescência para microtúbulo. Tamanho de mitocôndria, bactéria e cloroplasto COMPOSIÇÃO QUÍMICA MÉTODOS DE ESTUDO Membrana externa: 50% proteínas e 50% lipídios. Porina. Membrana interna: 80% proteínas e 20% lipídios Componentes da cadeia respiratória Succinato desidrogeanse ATP sintase Cardiolipina Matriz : DNA e RNA Ribonucleoproteínas e enzimas para a síntese protéica Enzimas das reações do ciclo de Krebs, da -oxidação dos ácidos graxos, do metabolismo dos compostos aminados. Enzimas para hidroxilação de intermediários da síntese de hormônios esteróides. DNA e RNA polimerase. Composição Química The structure of cardiolipin. Cardiolipin is an unusual lipid in the inner mitochondrial membrane. Mitochondrial and nuclear DNA stained with a fluorescent dye. This micrograph shows the distribution of the nuclear genome (red) and the multiple small mitochondrial genomes (bright yellow spots) in a Euglena gracilis cell. The DNA is stained with ethidium bromide, a fluorescent dye that emits red light. In addition, the mitochondrial matrix space is stained with a green fluorescent dye that reveals the mitochondria as a branched network extending throughout the cytosol. The superposition of the green matrix and the red DNA gives the mitochondrial genomes their yellow color. (Courtesy of Y. Hayashi and K. Ueda, J. Cell Sci. 93:565 570, 1989. © The Company of Biologists.) The production of mitochondrial and chloroplast proteins by two separate genetic systems. Most of the proteins in these organelles are encoded by the nucleus and must be imported from the cytosol. An electron micrograph of an animal mitochondrial DNA molecule caught during the process of DNA replication. The circular DNA genome has replicated only between the two points marked by red arrows. The newly synthesized DNA is colored yellow. (Courtesy of David A. Clayton.) ORIGEM Some major events that are believed to have occurred during the evolution of living organisms on earth. A suggested evolutionary pathway for the origin of mitochondria. Argumentos favoráveis à hipótese simbiótica 1. DNA mitocondrial é circular como o DNA das bactérias. 2. A síntese protéica das mitocôndrias é inibida por cloranfenicol como ocorre com as bactérias. 3. O coeficiente de sedimentação dos ribossomos de mitocôndrias (55S) é mais próximo de bactérias (70S) do que de eucariotos (80S). 4. A síntese protéica em mitocôndrias se inicia com formil-metionina como ocorre em bactérias. GENOMA, PROTEÍNAS E RNAs The organization of the human mitochondrial genome. The genome contains 2 rRNA genes, 22 tRNA genes, and 13 protein-coding sequences. The DNAs of many other animal mitochondrial genomes have also been completely sequenced. Most of these animal mitochondrial DNAs encode precisely the same genes as humans, with the gene order being identical for animals that range from mammals to fish. Electron micrographs of yeast cells. (A) The structure of normal mitochondria. (B) Mitochondria in a petite mutant. In petite mutants, all the mitochondrion-encoded gene products are missing, and the organelle is constructed entirely from nucleus-encoded proteins. (Courtesy of Barbara Stevens.) The origins of mitochondrial RNAs and proteins. The proteins encoded in the nucleus and imported from the cytosol have a major role in creating the genetic system of the mitochondrion, in addition to contributing most of the organelle's other proteins. Not indicated in this diagram are the additional nucleus-encoded proteins that regulate the expression of individual mitochondrial genes at posttranscriptional levels. The mitochondrion itself contributes only mRNAs, rRNAs, and tRNAs to its genetic system. FISIOLOGIA Simplified diagram of the three stages of cellular metabolism that lead from food to waste products in animal cells. This series of reactions produces ATP, which is then used to drive biosynthetic reactions and other energy-requiring processes in the cell. Stage 1 occurs outside cells. Stage 2 occurs mainly in the cytosol, except for the final step of conversion of pyruvate to acetyl groups on acetyl CoA, which occurs in mitochondria. Stage 3 occurs in mitochondria. An outline of glycolysis. Each of the 10 steps shown is catalyzed by a different enzyme. Note that step 4 cleaves a six-carbon sugar into two three-carbon sugars, so that the number of molecules at every stage after this doubles. As indicated, step 6 begins the energy generation phase of glycolysis, which causes the net synthesis of ATP and NADH molecules (see also Panel 2-8). The structure of the important activated carrier molecule acetyl CoA. A space-filling model is shown above the structure. The sulfur atom (yellow) forms a thioester bond to acetate. Because this is a high-energy linkage, releasing a large amount of free energy when it is hydrolyzed, the acetate molecule can be readily transferred to other molecules. How electrons are donated by NADH. In this diagram, the high-energy electrons are shown as two red dots on a yellow hydrogen atom. A hydride ion (H- a hydrogen atom and an extra electron) is removed from NADH and is converted into a proton and two high-energy electrons: H- H+ + 2e-. Only the ring that carries the electrons in a high-energy linkage is shown. Electrons are also carried ina similar way by FADH2. The path of electrons through the three respiratory enzyme complexes. The relative size and shape of each complex are shown. During the transfer of electrons from NADH to oxygen (red lines), ubiquinone and cytochrome c serve as mobile carriers that ferry electrons from one complex to the next. As indicated, protons are pumped across the membrane by each of the respiratory enzyme complexes. Quinone electron carriers. Ubiquinone in the respiratory chain picks up one H+ from the aqueous environment for every electron it accepts, and it can carry either one or two electrons as part of a hydrogen atom (yellow). When reduced ubiquinone donates its electrons to the next carrier in the chain, these protons are released. A long hydrophobic tail confines ubiquinone to the membrane and consists of 6 10 five-carbon isoprene units, the number depending on the organism. The corresponding electron carrier in the photosynthetic membranes of chloroplasts is plastoquinone, which is almost identical in structure. For simplicity, both ubiquinone and plastoquinone are referred to in this chapter as quinone (abbreviated as Q). The structure of the heme group attached covalently to cytochrome c. The porphyrin ring is shown in blue. There are five different cytochromes in the respiratory chain. Because the hemes in different cytochromes have slightly different structures and are held by their respective proteins in different ways, each of the cytochromes has a different affinity for an electron. Fosforilação Oxidativa Chemiosmotic coupling. Energy from sunlight or the oxidation of foodstuffs is first used to create an electrochemical proton gradient across a membrane. This gradient serves as a versatile energy store and is used to drive a variety of energy-requiring reactions in mitochondria, chloroplasts, and bacteria. The general mechanism of oxidative phosphorylation. (A) As a high-energy electron is passed along the electron-transport chain, some of the energy released is used to drive the three respiratory enzyme complexes that pump H+ out of the matrix. The resulting electrochemical proton gradient across the inner membrane drives H+ back through the ATP synthase, a transmembrane protein complex that uses the energy of the H+ flow to synthesize ATP from ADP and Pi in the matrix. (B) An electron micrograph of the inside surface of the inner mitochondrial membrane in a plant cell. Densely packed particles are visible, due to protruding portions of the ATP synthases and the respiratory enzyme complexes. (Micrograph courtesy of Brian Wells.) Hipótese Quimiosmótica (Peter Mitchell, 1961) A membrana interna da mitocôndria precisa estar intacta para que a fosforilação oxidativa ocorra. Os carreadores de elétrons da cadeia respiratória servem como um sistema de transporte ativo para transportar prótons da matriz para o espaço intermembranas, gerando um gradiente de prótons. O gradiente de prótons gerado, é usado para sintetizar ATP. A general model for H+ pumping. This model for H+ pumping by a transmembrane protein is based on mechanisms that are thought to be used by both cytochrome oxidase and bacteriorhodopsin. The protein is driven through a cycle of three conformations: A, B, and C. As indicated by their vertical spacing, these protein conformations have different energies. In conformation A, the protein has a high affinity for H+, causing it to pick up a H+ on the inside of the membrane. In conformation C, the protein has a low affinity for H+, causing it to release a H+ on the outside of the membrane. The transition from conformation B to conformation C that releases the H+ is energetically unfavorable, and it occurs only because it is driven by being allosterically coupled to an energetically favorable reaction occurring elsewhere on the protein (blue arrow). The ATP synthase is a reversible coupling device that can convert the energy of the electrochemical proton gradient into chemical-bond energy, or vice versa. The ATP synthase can either (A) synthesize ATP by harnessing the proton-motive force or (B) pump protons against their electrochemical gradient by hydrolyzing ATP. ATP synthase. (A) The enzyme is composed of a head portion, called the F1 ATPase, and a transmembrane H+ carrier, called F0. Both F1 and F0 are formed from multiple subunits, as indicated. A rotating stalk turns with a rotor formed by a ring of 10 to 14 c subunits in the membrane (red). The stator (green) is formed from transmembrane a subunits, tied to other subunits that create an elongated arm. This arm fixes the stator to a ring of 3a and 3b subunits that forms the head. (B) The three-dimensional structure of the F1 ATPase, determined by x-ray crystallography. This part of the ATP synthase derives its name from its ability to carry out the reverse of the ATP synthesis reaction namely, the hydrolysis of ATP to ADP and Pi, when detached from the transmembrane portion. Nature 370:621 628, 1994. The binding-change mechanism of ATP synthesis from ADP and Pi by the F0F1 complex. The three subunits alternate between three conformational states that differ in their binding affinities for ATP, ADP, and Pi. Step 1 : After ADP and Pi bind to one of the three subunits (here, arbitrarily designated 1) whose nucleotide-binding site is in the O (open) conformation, proton flux powers a 120° rotation of the g subunit (relative to the fixed subunits). This causes an increase in the binding affinity of the 1 subunit for ADP and Pi to L (low), an increase in the binding affinity of the 3 subunit for ADP and Pi from L to T (tight), and a decrease in the binding affinity of the 2 subunit for ATP from T to O, causing release of the bound ATP. Step 2 : The ADP and Pi in the T site (here the 3 subunit) form ATP, a reaction that does not require an input of energy, and ADP and Pi bind to the 2 subunit, which is in the O state. This generates an F1 complex identical with that which started the process (upper left) except that it is rotated 120°. Step 1 now occurs again, and the cycling of the O L → T → O conformations of each subunit continues. [Adapted from P. Boyer, 1989, FASEB J. 3:2164, and Y. Zhou et al., 1997, Proc. Nat'l. Acad. Sci. USA 94:10583.] Rendimento de ATP na mitocôndria • De glicose até Piruvato: 2 moléculas de ATP • Cada NADH que cede 1 par de elétrons na cadeia respiratória rende em média 2,5 moléculas de ATP. • Cada FADH2 que cede um par de elétrons na cadeia respiratória rende em média 1,5 molécula de ATP. • Cada 2 moléculas de Piruvato que entram mitocôndria produzem 2 acetil CoA que geram 2 NADH, que ao cederem elétrons na cadeia respiratória produzem 5 moléculas de ATP • Cada acetil-CoA que entra no Ciclo de Krebs produz uma molécula de GTP que logo se transforma em ATP. • Como no ciclo de Krebs são produzidos 3 NADH e 1 FADH2 e 1 ATP para cada Piruvato/Acetil CoA, teremos uma produção de (3x2,5) + (1x1,5) + 1 = 10 moléculas de ATP. Como são dois Acetil CoA, então a produção de ATP é de 20 moléculas de ATP. • Considerando os elétrons provenientes das 2 moleçulas de NADH produzidas na glicólise , teremos mais 2x2,5 moléculas de ATP, mais o rendimento de 2 moleculas de ATP na glicólise. • Assim para cada molécula de glicose podem ser produzidas 32 moléculas de ATP. Some of the active transport processes driven by the electrochemical proton gradient across the inner mitochondrialmembrane. Pyruvate, inorganic phosphate (Pi), and ADP are moved into the matrix, while ATP is pumped out. The charge on each of the transported molecules is indicated for comparison with the membrane potential, which is negative inside, as shown. The outer membrane is freely permeable to all of these compounds. Entrada de ácido graxo na mitocôndria Síntese de hormônio esteróide Doenças mitocondriais Neuropatia optica hereditária de Leber Devido mutação no DNA mitocondrial , afeta o complexo I (NADH desidrogenase, substitui uma His por Arg) do sistema de transporte de elétrons. Outra mutação que também afeta esse complexo pode causar desordem muscular. (Cooper – The Cell) Epilepsia mioclonica e doença da fibra vermelha irregular (MERRF). Causada por uma mutação em um dos genes que produzem RNAt mitocondrial, levando a uma diminuiç ão na síntese de proteínas mitocondriais responsáveis pelo transporte de elétrons e produção de ATP. Resumo de Mitocôndria 1. Não podem ser vistas com detalhes no microscópio ótico. 2. Tem duas membranas sendo que a interna é bem mais seletiva do que a externa. 3. Tem DNA, RNA e ribossomos. 4. Origem: Teoria Simbiótica. 5. Funções: Respiração celular aeróbica, síntese de ATP, β-oxidação de ácidos graxos, participa da síntese de hormônios esteróides, participa do ciclo da uréia. 6. Piruvato Desidrogenase: Piruvato é descaboxilado e desidrogenado gerando Acetil-Coa. 7. Ciclo de Krebs: Acetil-Coa entra no ciclo de Krebs. Várias reações ocorrem resultando em 3 NADH + H+ e 1 FADH2 , 1 ATP e 2 descarboxilações, para cada acetil-CoA que entra no ciclo de Krebs. 8. NADH + H+ e FADH2 cedem seus elétrons para a cadeia respiratória resultando em ejeção de prótons e redução de O2 que então resulta em formação de água. 9. Fosforilação oxidativa do ADP formando ATP. 10. β-oxidação de ácidos graxos resulta em liberação de Acetil-CoA, que entra no ciclo de Krebs. 11. Rendimento de produção de ATP por molécula de glicose: 30 ou 32 moléculas de ATP.
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