CHAPTER 5 Aerobic Respiration and the Mitochondrion Introduction

CHAPTER 5 Aerobic Respiration and the Mitochondrion

Introduction • The early Earth was populated by anarobes, which captured and utilized energy by oxygenindependent metabolism. • Oxygen accumulated in the primitive atmosphere after cyanobacteria appeared. • Aerobes evolved to use oxygen to extract more energy from organic molecules. • In eukaryotes, aerobic respiration takes place in the mitochondrion.

5. 1 Mitochondrial Structure and Function (1) • Mitochondria have characteristic morphologies despite variable appearance. – Typical mitochondria are bean-shaped organelles but may be round or threadlike. – The size and number of mitochondria reflect the energy requirements of the cell.

Mitochondria

Mitochondrial Structure and Function (2) • Mitochondria can fuse with one another, or split in two. – The balance between fusion and fission is likely a major determinant of mitochondrial number, length, and degree of interconnection.

Mitochondrial fusion and fission

Mitochondrial Structure and Function (3) • Inner and outer mitochondrial membranes enclose two spaces: the matrix and intermembrane space. – The outer mitochondrial membrane serves as its outer boundary. – The inner mitochondrial membrane is subdivided into two interconnected domains: • Inner boundary membrane • Cristae – where the machinery for ATP is located

The structure of a mitochondrion

Mitochondrial Structure and Function (4) • Mitochondrial Membranes – The outer membrane is about 50%; the inner membrane is more than 75% protein. – The inner membrane contains cardiolipin but not cholesterol, both are true of bacterial membranes. – The outer membrane contains a large poreforming protein called porin. – The inner membrane is impermeable to even small molecules; the outer membrane is permeable to even some proteins.

Porins

Mitochondrial Structure and Function (5) • The mitochondrial matrix – Contains a circular DNA molecule, ribosomes, and enzymes. – RNA and proteins can be synthesized in the matrix.

Overview of carbohydrate metabolism in eukaryotic cells

5. 2 Oxidative Metabolism in the Mitochondrion (1) • The first steps in oxidative metabolism are carried out in glycolysis. – Glycolysis produces pyruvate, NADH, and two molecules of ATP. – Aerobic organisms use O 2 to extract more than 30 additional ATPs from pyruvate and NADH. – Pyruvate is transported across the inner membrane and decarboxylated to form acetyl Co. A, which enters the next stage.

An overview of glycolysis

Oxidative Metabolism in the Mitochondrion (2) • The tricarboxylic acid (TCA) cycle – It is a stepwise cycle where substrate is oxidized and its energy conserved. – The two-carbon acetyl group from acetyl Co. A is condensed with the four-carbon oxaloacetate to form a six-carbon citrate. – During the cycle, two carbons are oxidized to CO 2, regenerating the four-carbon oxaloacetate needed to continue the cycle.

The TCA cycle

Oxidative Metabolism in the Mitochondrion (3) • The TCA cycle (continued) – Four reactions in the cycle transfer a pair of electrons to NAD+ to form NADH, or to FAD+ to form FADH 2. – Reaction intermediates in the TCA cycle are common compounds generated in other catabolic reactions making the TCA cycle the central metabolic pathway of the cell.

Catabolic pathways generate compounds that are fed into the TCA cycle

Oxidative Metabolism in the Mitochondrion (4) • The Importance of Reduced Coenzymes in the Formation of ATP – The reduced coenzymes FADH 2 and NADH are the primary products of the TCA cycle. – NADH formed during glycolysis enters the mitochondria via malate-aspartate or glycerol phosphate shuttles.

The glycerol phosphate shuttle

Oxidative Metabolism in the Mitochondrion (5) • The Importance of Reduced Coenzymes – As electrons move through the electron-transport chain, H+ are pumped out across the inner membrane. – ATP is formed by the controlled movement of H+ back across the membrane through the ATPsynthesizing enzyme.

Oxidative Metabolism in the Mitochondrion (6) • Reduced coenzymes (continued) – The coupling of H+ translocation to ATP synthesis is called chemiosmosis. – Three molecules of ATP are formed from each pair of electrons donated by NADH; two molecules of ATP are formed from each pair of electrons donated by FADH 2.

Summary of oxidative phosphorylation

The Human Perspective: The Role of Anaerobic and Aerobic Metabolism in Exercise (1) • ATP hydrolysis increases 100 -fold during exercise, quickly exhausting ATP available. • Muscles used stored creatine phosphate (Cr. P) to rapidly generate but must rely on aerobic or anaerobic synthesis of new ATP for sustained activity. Cr. P + ADP Cr + ATP

The Human Perspective: The Role of Anaerobic and Aerobic Metabolism in Exercise (2) • Fast-twitch muscle fibers contract rapidly, have few mitochondria ad produce ATP anaerobically. – Anaerobic metabolism produces fewer ATPs per glucose but produces them very fast. – Anaerobic metabolism rapidly depletes available glucose and builds up lactic acid which reduces cellular p. H.

Skeletal muscles

The Human Perspective: The Role of Anaerobic and Aerobic Metabolism in Exercise (3) • Slow-twitch fibers contract slowly, have many mitochondria and produce most of their ATP by aerobic metabolism. – Aerobic metabolism initially uses glucose as a substrate. – Free fatty acids are oxidized during prolonged exercise. • Ratio of fast- to slow-twitch fibers is variable and depends on the normal function of the muscle.

5. 3 The Role of Mitochondria in the Formation of ATP (1) • ATP can be formed by substrate-level phosphorylation or oxidative phosphorylation. – Accounts for > 160 kg of ATP in our bodies per day

The Role of Mitochondria in the Formation of ATP (2) • Oxidation-Reduction (Redox) Potentials – Strong oxidizing agents have a high affinity for electrons; strong reducing agents have a weak affinity for electrons – Redox reactions are accompanied by a decrease in free energy. – The transfer of electrons causes charge separation that can be measured as a redox potential.

Redox potential of some reaction couples

The Role of Mitochondria in the Formation of ATP (3) • Electron Transport – Electrons move through the inner membrane via a series of carriers of decreasing redox potential. – Electrons associated with either NADH or FADH 2 are transferred through specific electron carriers that make up the electron transport chain.

The Role of Mitochondria in the Formation of ATP (4) • Types of Electron Carriers – Flavoproteins are polypeptides bound to either FAD or FMN. – Cytochromes contain heme groups bearing Fe or Cu metal ions. – Three cooper atoms are located within a single protein complex and alternate between Cu 2+/Cu 3+ – Ubiquinone (coenzyme Q) is a lipid-soluble molecule made of five-carbon isoprenoid units.

Structures of three electron carriers

The Role of Mitochondria in the Formation of ATP (5) • Types of Electron Carriers (continued) – Iron-sulfur proteins contain Fe in association with inorganic sulfur. – These carriers are arranged in order of increasingly positive redox potential. – Sequence of carriers determined by use if inhibitors.

Sequence of electron carriers

The Role of Mitochondria in the Formation of ATP (6) • Electron-Transport Complexes – Complex I (NADH dehydrogenase) catalyzes transfer of electrons from NADH to ubiquinone and transports four H+ per pair. – Complex II (succinate dehydrogenase) catalyzes transfer of electrons from succinate to FAD to ubiquinone without transport of H+. – Complex III (cytochrome bc 1) catalyzes the transfer of electrons from ubiquinone to cytochrome c and transports four H+ per pair.

The electron-transport chain of the inner mitochondrial membrane

The electron-transport chain of the inner mitochondrial membrane

The Role of Mitochondria in the Formation of ATP (7) • Electron-Transport Complexes (continued) – Complex IV (cytochrome c oxidase) catalyzes transfer of electrons to O 2 and transports H+ across the inner membrane. – Cytochrome oxidase is a large complex that adds four electrons to O 2 to form two molecules of H 2 O. – The metabolic poisons CO, N 3–, and CN– bind catalytic sites in Complex IV.

Cytochrome oxidase

The Role of Mitochondria in the Formation of ATP (8) • Cytochrome oxidase – Electrons are transferred one at a time. – Energy released by O 2 reduction is presumably used to drive conformational changes. – These changes would promote the movement of H+ ions and through the protein.

5. 4 Translocation of Protons and the Establishment of a Proton-Motive Force (1) • There are two components of the proton gradient: – The concentration gradient between the matrix and intermembrane space creates a p. H gradient (Δp. H). – The separation of charge across the membrane creates an electric potential (Ψ). – The energy present in both components of the gradients is proton-motive force (Δp).

Visualizing the proton-motive force

Translocation of Protons and the Establishment of a Proton-Motive Force (2) • Dinitrophenol (DNP) uncouples glucose oxidation and ATP formation by increasing the permeability of the inner membrane to H+, thus eliminating the proton gradient. • Differences in uncoupling proteins (UCPs) account for differences in metabolic rate.

5. 5 The Machinery for ATP Formation (1) • Isolation of coupling factor 1, or F 1, showed that it hydrolyzed ATP. – Under experimental conditions, it behaves as an ATP synthase. – Led to conclusion that an ionic gradient establishes a protonmotive force to phosphorylate ADP.

An experiment to drive ATP formation in membrane vesicles reconstituted with the Na+/K+-ATPase

The Machinery for ATP Formation (2) • The structure of the ATP synthase: – The F 1 particle is the catalytic subunit, and contains three catalytic sites for ATP synthesis. – The F 0 particle attaches to the F 1 and is embedded in the inner membrane. – The F 0 base contains a channel through which protons are conducted from the intermembrane space to the matrix–demonstrated in experiments with submitochondrial particles.

The structure of the ATP synthase

ATP formation in experiments with submitochondrial particles

The Machinery for ATP Formation (3) • The Basis of ATP Formation According to the Binding Change Mechanism – The binding change mechanism states the following: • Movement of protons through ATP synthase alters the binding affinity of the active site. • Each active site goes through distinct conformations that have different affinities for substrates and product.

The structural basis of catalytic site conformation

The binding change mechanism for ATP synthesis

The Machinery for ATP Formation (4) • Binding change mechanism – Binding sites on the catalytic subunit can be tight, loose, or open. – ATP is synthesized through rotational catalysis where the stalk of ATP synthase rotates relative to the head. – There is structural and experimental evidence to support this mechanism

Direct observation of rotational catalysis

The Machinery for ATP Formation (5) • Using the Proton Gradient to Drive the Catalytic Machinery: The Role of the F 0 Portion of ATP Synthase – The c subunits of the F 0 base form a ring. – The c ring is bound to subunit of the stalk. – Protons moving through membrane rotate the ring. – Rotation of the ring provides twisting force that drives ATP synthesis.

A model of the proton diffusion coupled to rotation of c ring in the F 0 complex

The Machinery for ATP Formation (6) • Other Roles for the Proton-Motive Force in Addition to ATP Synthesis – The H+ gradient drives transport of ADP into and ATP out of the mitochondrion. – ADP is the most important factor controlling the respiration rate. – Many factors influence the rate of respiration, but the pathways are poorly understood.

Summary of the major activities during aerobic respiration in the mitochondrion

5. 6 Peroxisomes (1) • Peroxisomes are membrane-bound vesicles that contain oxidative enzymes. • They oxidize very-long-chain fatty acids, and synthesize plasmalogens (a class of phospholipids). • They form by splitting from preexisting organelles, import preformed proteins, and engage in oxidative metabolism.

The structure and function of peroxisomes

Peroxisomes (2) • Hydrogen peroxide (H 2 O 2), a reactive and toxic compound, is formed in peroxisomes and is broken down by the enzyme catalase. • Plants contain a special peroxisome called glyoxysome, which can convert fatty acids to glucose by germinating seedlings.

Glyoxysome localization within plant seedlings

The Human Perspective: Diseases that Result from Abnormal Mitochondrial or Peroxisomal Function (1) • Mitochondria – A variety of disorders are known that result from abnormalities in mitochondria structure/function. – Majority of mutations linked to mitochondrial diseases are traced to mutations in mt. DNA. – Mitochondrial disorders are inherited maternally.

Mitochondrial abnormalities in skeletal muscle

The Human Perspective: Diseases that Result from Abnormal Mitochondrial or Peroxisomal Function (2) • It is speculated that accumulations of mutations in mt. DNA is a major cause of aging. • In mice encoding a mutation in their mt. DNA, signs of premature aging develop. • Additional findings suggest that mutations in mt. DNA may cause premature aging but are not sufficient for the normal aging process.

A premature aging phenotype caused by mutations in mt. DNA

The Human Perspective: Diseases that Result from Abnormal Mitochondrial or Peroxisomal Function (3) • Peroxisomes – Patients with Zellweger syndrome lack peroxisomal enzymes due to defects in translocation of proteins from the cytoplasm into the peroxisome. – Adrenoleukodydstrophy is caused by lack of a peroxisomal enzyme, leading to fatty acid accumulation in the brain and destruction of the myelin sheath of nerve cells.