Components and Roles of ATP Synthase Complex in Oxidative Phosphorylation, Study notes of Biology

Download Components and Roles of ATP Synthase Complex in Oxidative Phosphorylation and more Study notes Biology in PDF only on Docsity! Oxidative Phosphorylation -1 Biochemistry 460 - Dr. Tischler OXIDATIVE PHOSPHORYLATION Related Reading: Chapter 18: 520-524; 527-534 in Stryer 6th edition OBJECTIVES: 1. Distinguish between electroneutral and electrogenic transport. 2. Describe the significance of electrogenic transport for the adenine nucleotide transporter and the role of this transporter. 3. List the features of the chemiosmotic model. 4. Name the components of the ATP synthase complex, and describe their roles. 5. Discuss how the malate-aspartate and the α-glycerol phosphate differ. (Do not memorize the layout of the shuttle to reproduce it but understand their key aspects) 6. Define respiratory control and uncoupling, and describe the physiological importance of these processes. PHYSIOLOGICAL PREMISE Why do some snake and spider venoms cause cell death at the site of the bite in particular? Some of these venoms contain enzymes called phospholipases. Phospholipases hydrolyze membrane phospholipids to release fatty acids. Phospholipases in snake or spider venom degrade phospholipids in the mitochondrial membrane. The fatty acids released act as natural uncouplers that, as is described in this lecture, prevent oxidative phosphorylation by destroying the pH gradient. Consequently the cell dies because of an inability to produce enough energy. MITOCHONDRIAL TRANSPORT Electroneutral Transport Because the mitochondrial inner membrane is highly impermeable, it contains many transport proteins to control the movement of substances into and out of the matrix. Most substances move down their concentration gradient. Thus when citrate is produced in large amounts in the matrix for fatty acid or cholesterol synthesis, citrate generates a large concentration difference, which favors its movement to the cytoplasm where it can participate in these pathways. Most of the transport systems require the exchange of molecules, and most of these exchanges occur with molecules having the same charge; this is termed electroneutral transport. For instance pyruvate (-1 charge), which enters the matrix for further metabolism via pyruvate dehydrogenase or pyruvate carboxylase, exchanges with a negatively charged hydroxyl ion (OH-). Phosphate (PO4-), which enters the matrix for synthesis of ATP (see Fig. 3), also can exchange with OH-. Citrate, a tricarboxylic acid, has a negative 3 (3-) charge at physiological pH, but exchanges with malate, a dicarboxylic acid (2-). To maintain electroneutrality of this exchange, a proton accompanies the citrate thus neutralizing one of its negative charges (citrate3- + H+ swaps with malate2-). Electrogenic Transport There are some transport systems in which the exchange of molecules involves unequal charges. When there is the net movement of charge across the membrane, this is termed electrogenic transport. In mitochondria, net negative charges move out of the matrix while net positive charges must move into the matrix by following the charge gradient. Recall that the pumping of protons creates a gradient of negative inside to positive outside so that the more negatively charged matrix will favor the net outward movement of negative charge. Oxidative Phosphorylation -2 Figure 1. Electrogenic transport system in mitochondria. The adenine nucleotide exchange ensures that ATP4- produced in the mitochondrial matrix is transported to the cytoplasm where it is needed. The exchange if aspartate (in) for glutamate (out) is unique because both have a negative one charge however a proton accompanies the glutamate to effectively neutralize its charge creating electrogenic exchange. The most important electrogenic transporter is the adenine nucleotide transporter. ATP produced in the mitochondrial matrix by oxidative phosphorylation (see Fig. 2) is needed in the cytoplasm for energy- requiring processes such as muscle contraction, lipogenesis, cholesterol synthesis and gluconeogenesis. Hence, it is obligatory that ATP only be transported into the cytoplasm while ADP moves into the mitochondria matrix where it can be phosphorylated to ATP. ADP bears a negative three charge whereas ATP has a negative four charge at physiological pH (Fig. 1). Thus, the exchange of ATP4- moving out with ADP3- moving in creates a net charge of negative one moving into the intermembrane space. The charge gradient facilitates this exchange of ADP for ATP. The reverse exchange cannot occur in functional mitochondria because it would require the more negative ATP molecule to move towards the more negative environment of the matrix, a process that cannot occur Similarly the glutamate-aspartate translocase is electrogenic because glutamate1− outside is neutralized by cotransport of a H+ into the matrix in exchange for aspartate1− moving out. This translocase is an integral part of the malate-aspartate shuttle (described later in this lecture). Electrogenic translocases are irreversible as long as a charge gradient exists across the membrane (more negative inside because the protons pumped out are positive). Electrogenic transport requires energy but ensures unidirectional outward flux of ATP and aspartate under normal circumstances. OXIDATIVE PHOSPHORYLATION – THE CHEMIOSMOTIC MODEL: Overview Oxidative phosphorylation consists of two processes. The oxidative portion is the respiratory chain and is recreated in the right hand side of fig 2. Shown is the movement of protons through complexes I, III and IV creating a proton and charge gradient across the inner mitochondrial membrane. This gradient is then used to provide energy to produce ATP, the phosphorylation part of the process (lower left of Fig 2; described below). The charge gradient facilitates the inward transport of ADP and the efflux of ATP (upper left of Fig. 2; described later in this lecture). INNER MEMBRANE Matrix Side Intermembrane Space Aspartate1- Glutamate1- + H+ Aspartate translocase Electrogenic Translocases ADP3- ATP4- Adenine nucleotide translocase Oxidative Phosphorylation -5 Figure 3. The malate-aspartate shuttle. Cytoplasmic malate dehydrogenase (1) reduces oxaloacetate (OAA) to malate. The α-ketoglutarate (KG) transporter (2) exchanges malate for KG. Mitochondrial malate dehydrogenase (3) generates intramitochondrial NADH by oxidation of malate to oxaloacetate. Mitochondrial aspartate aminotransferase (4) catalyzes the transfer of an amino group from glutamate (glu) to oxaloacetate to produce KG and aspartate (asp). KG is transported out on its translocase (2) and aspartate is transported out on the unidirectional aspartate translocase (5). Cytoplasmic aspartate aminotransferase (6) regenerates oxaloacetate for reaction (1) by transferring the amino group from aspartate to KG producing glutamate, which is transported into the matrix in exchange for aspartate (5). Glycerol Phosphate Shuttle (Figure 4) The glycerol phosphate shuttle occurs almost exclusively in the liver. Electrons from cytoplasmic NADH reduce dihydroxyacetone phosphate to glycerol phosphate, which in turn carries the electrons to the respiratory chain. Electrons reach the respiratory chain via glycerol phosphate dehydrogenase that is bound to the inner membrane. This enzyme contains a FAD prosthetic group, as we showed for succinic dehydrogenase of the citric acid cycle. Thus, electrons are fed directly to coenzyme Q. Since complex I is bypassed, this shuttle produces one fewer ATP from glycolytic NADH than occurs with the malate- aspartate shuttle. Also unlike the other shuttle, the electron carrier, glycerol 3-phosphate, never permeates the inner membrane but interacts instead with the transmembrane glycerol 3-phosphate dehydrogenase. NADH OAA Malate KG Glu0 Asp1- KG OAA NADH NAD+Malate (1) (3) (4) (5) (2) (6) OAA = Oxaloacetate Glu = glutamate Asp = aspartate KG = α-ketoglutarate Glucose Pyruvate CYTOPLASM MATRIX GLYCOLYSIS Complex I e- e- =electrons OUTER MEMBRANE NAD+ NAD+ INNER MEMBRANE e- e- e- e- e- Asp1- Glu0 Oxidative Phosphorylation -6 Figure 4. Glycerol phosphate shuttle. Cytoplasmic glycerol 3-phosphate dehydrogenase (1) oxidizes NADH. Glycerol 3-phosphate dehydrogenase in the inner mitochondrial membrane (2) reduces bound FAD to FADH2. CONTROL OF MITOCHONDRIAL RESPIRATION (OXYGEN CONSUMPTION) Respiratory Control The rate of respiration is ultimately controlled by the availability of ADP in the mitochondrial matrix. This is referred to as respiratory control. ADP binding to the F1, signals the stalk to open the proton channel. Since inorganic phosphate is abundant, the concentration of ADP determines when ATP is to be synthesized. When energy demands are high the concentration of ADP will be high in this cell. The elevated concentration of ADP in the mitochondrial matrix will promote the inward movement of protons for ATP synthesis. As protons are transferred inward down their gradient to support ATP synthesis, the respiratory chain responds by pumping out more protons in conjunction with an increased rate of respiration (O2 consumption). In this way ADP provides control of respiration. When the concentration of ADP is low, such as occurs at rest, respiration slows because the pH gradient reaches a maximum amount and more protons cannot be pumped out. In the presence of inhibitors of the respiratory chain, respiratory control is lost because protons can no longer be pumped out even when the concentration of ADP increases. Dihydroxyacetone phosphate (DHAP) NADH DHAP G3P FAD FADH2 CoQ Glycerol 3-phosphate dehydrogenase (1) Glycerol 3-phosphate (2) O2 MATRIX e- =electrons e- e- e- e- e- Glucose Pyruvate GLYCOLYSIS NAD+ NAD+ CYTOPLASM OUTER MEMBRANE INNER MEMBRANE Oxidative Phosphorylation -7 Uncoupling (Figure 5) Figure 5. The effects of uncoupling on glucose, lactate and mitochondrial metabolism. Uncouplers are substances (e.g., natural uncoupling proteins, fatty acids or chemicals such as dinitrophenol {DNP}) that bind protons and are hydrophobic. Because of their hydrophobicity, these substances can diffuse across the inner mitochondrial membrane, despite its relative impermeability. By carrying protons into the matrix, uncouplers destroy (collapse or dissipate) the pH gradient by equilibrating the concentration of protons across the membrane. This causes ATP formation to cease because there is no longer an existing pH gradient to drive ATP synthesis. However, oxygen consumption remains rapid because the respiratory chain continues to attempt to maintain a pH gradient, albeit unsuccessfully. Remember that the rate of the respiratory chain actually responds to the size of the pH gradient; the smaller the gradient the faster the rate. Thus artificial dissipation of the gradient creates a constant rapid rate of respiration. The glycolytic pathway will operate rapidly in conjunction with the malate-aspartate shuttle quickly oxidizing any NADH that forms. Pyruvate will be preferentially oxidized to CO2 rather than reduced to lactate because NADH amounts are low. ALANINE GLYCOGEN CYTOPLASM INTERMEMBRANE SPACE LACTATE NADH MITOCHONDRIAL MATRIX H+ ATP ADP + Pi ATP ADP Pi NADH MALATE-ASP SHUTTLE PYRUVATE GLUCOSE PYRUVATE ACETYL CoA PDH PC TCA CYCLE SDH complex I NADH NAD complex III complex IV CS KgDH MDH H+ OAA Malate ICDH H+ X uncoupler