11 Bioenergetics and Metabolism Chapter Outline: Mitochondria Oxidative Phosphorylation Chloroplasts and Other Plastids Photosynthesis Peroxisomes amyloplast

Mitochondria, chloroplasts, peroxisomes Student learning outcomes: Explain similarities, differences structure and function of mitochondria, chloroplast, peroxisome Explain process of transport of proteins to organelles: signals on proteins, complexes that assist Explain metabolic functions of mitochondria, chloroplast: membrane compartments, proton gradient and ATP Mitochondria and chloroplasts have genomes

Figure 10.3** Overview of protein sorting Cell5e-Fig-10-03-0.jpg Fig. 10.3

Generation of metabolic energy- major cell activity Introduction Generation of metabolic energy- major cell activity Mitochondria generate energy from breakdown of lipids and carbohydrates. Chloroplasts use sunlight energy to generate ATP and the reducing power needed to synthesize carbohydrates from CO2 and H2O. Peroxisomes contain metabolic enzymes: fatty acid oxidation, generate peroxides, have catalase

Mitochondria are surrounded by double membrane: Outer membrane permeable to small molecules Inner membrane has numerous folds (cristae); extend into interior (matrix). Fig. 11.1

Fig 11.2 Metabolism in the matrix of mitochondria Matrix contains small genome (human 17 kb; yeast 80 kb) Enzymes for oxidative metabolism: Pyruvate (from glycolysis) into mitochondria; complete oxidation to CO2 yields most of energy (ATP) from glucose Enzymes of citric acid (Krebs) cycle - in mitochondrial matrix. Most of energy produced by oxidative phosphorylation, occurs on inner mitochondrial membrane (electron transport chain) Fig. 11.2 Cell5e-Fig-11-02-0.jpg

Mitochondria High-energy electrons from NADH and FADH2 transferred through a membrane carriers membrane to molecular oxygen Energy of electrons converted to potential energy stored in a proton gradient, which drives ATP synthesis. Inner membrane has many proteins involved in oxidative metabolism and transport Inner membrane impermeable to most ions, small molecules

Outer mitochondrial membrane highly permeable to small molecules: Porins form channels for free diffusion of small molecules. Composition of intermembrane space similar to cytosol (with pH ~7; matrix pH ~8) Mitochondria can fuse, also can divide

Genomes reflect endosymbiotic origin: Mitochondria have DNA Genomes reflect endosymbiotic origin: usually circular DNA molecules, multiple copies. encode only a few proteins (some oxidative phosphorylation). encode rRNAs and most tRNAs needed for translating protein-coding sequences Ribosomes are in matrix Some different codon usage Table11.1 Human mtDNA 16-kb Fig. 11.3

Mutations in mitochondrial genes cause disease Molecular Medicine 11.1 Diseases of Mitochondria: Leber’s Hereditary Optic Neuropathy: LHON mutations in mitochondrial DNA Mutations in mitochondrial genes cause disease Leber’s hereditary optic neuropathy, blindness; mutations in mitochondrial genes: components of electron transport chain Cell5e-MM-11-01-0.jpg

Genes for many mitochondrial proteins in nucleus. Some genes transferred from prokaryotic ancestor Most proteins are synthesized on free cytosolic ribosomes, imported to mitochondria as complete polypeptides. Because of double-membrane structure of mitochondria, import of proteins is complex Matrix proteins are targeted by NH2-terminal sequences (presequences); removed after import

Figure 11.4 Import of mitochondrial matrix proteins Membrane or free proteins Presequences target Tom receptors/ channels on outer membrane (translocase) Tim receptors on inner membrane Electrochemical gradient Hsp70 Chaperones MPP cleavage ATP hydrolysis Compare ER/Golgi Cell5e-Fig-11-04-0.jpg Fig. 11.4

Figure 11.5 Binding cycle of an Hsp70 chaperone Presequence cleaved by matrix processing peptidase (MPP) Hsp70 chaperones facilitate folding. Similarity to signal peptidase for ER Cell5e-Fig-11-05-0.jpg Fig. 11.5

Inner membrane proteins are small molecule transporters. Figure 11.6 Import of small molecule transport proteins into the mitochondrial inner membrane Inner membrane proteins are small molecule transporters. multiple internal import signals, Hsp90 chaperone , plusTom70, translocates across channel. Intermembrane: proteins escorted by mobile Tim22, “Tiny Tims”. Translocated through Tim22; internal stop-transfer signals causes exit insert inner membrane. Cell5e-Fig-11-06-0.jpg Fig. 11.6

Both presequences, internal signal sequences. Figure 11.7 Sorting of proteins containing presequences to different mitochondrial compartments Both presequences, internal signal sequences. Translocated in Tom40. Some exit channel laterally, Some remain in intermembrane space Others transported back to intermembrane space Or inserted into inner membrane Cell5e-Fig-11-07-0.jpg Fig. 11.7

Outer membrane proteins: Figure 11.8 Insertion of β-barrel proteins into the mitochondrial outer membrane Outer membrane proteins: including Tom40 and β-barrel proteins (e.g., porins), Pass through Tom complex into intermembrane space. Carried by Tiny Tims to a SAM (sorting and assembly machinery) complex Inserted into outer membrane Cell5e-Fig-11-08-0.jpg Fig. 11.8

Phospholipids are imported from cytosol. Mitochondria Phospholipids are imported from cytosol. Phospholipid transfer proteins: take phospholipids from ER membrane, transport them through cytosol, released at new membrane (e.g. mitochondria) Mitochondria catalyze synthesis of cardiolipin Phospholipid with four fatty acid chains..

Figure 10.3** Overview of protein sorting Cell5e-Fig-10-03-0.jpg

The Mechanism of Oxidative Phosphorylation Electrons from NADH and FADH2 combine with O2: Energy released from oxidation/reduction reactions drives ATP synthesis Electrons travel through electron transport chain Proteins on inner mitochondrial membrane Sets up proton gradient across membrane Intermembrane space has lower pH (more H+) Chemiosmotic mechanism for synthesis of ATP: Protons returning to matrix power ATP synthase.

Fig 11.10 Transport of electrons from NADH Transfer of electrons from NADH: Complex I, Coenzyme Q (ubiquinone) Complex III Cytochrome c Complex IV (cytochrome oxidase) to O2 3 H+ transported across membrane V is ATP synthase: H+ reentry gives ATP Cell5e-Fig-11-10-0R.jpg Fig. 11.10

Fig 11.11 Transport of electrons from FADH2 Transfer of electrons from FADH2: Complex II (less energy) Coenzyme Q (ubiquinone) Complex III Cytochrome c Complex IV (cytochrome oxidase) to O2 3 H+ transported across membrane V is ATP synthase: H+ reentry gives ATP Cell5e-Fig-11-11-0.jpg Fig. 11.11

The Mechanism of Oxidative Phosphorylation Chemiosmotic coupling mechanism: Couples electron transport to ATP generation. Electron transport coupled to transport of protons to intermembrane space Proton gradient across inner membrane Also electric potential Electrochemical gradient exists Fig. 11.12

Fig 11.13 Structure of ATP synthase Phospholipid bilayer impermeable to ions Protons cross through protein channel. Energy converted to ATP in complex V (ATP synthase): F0 is channel F1 rotates, makes ATP 4 protons to synthesize 1 ATP: 1 NADH yields 3 ATP; 1 FADH2 yields 2 ATP Cell5e-Fig-11-13-0.jpg Fig. 11.13

Fig 11.14 Transport of metabolites across the mitochondrial inner membrane Electrochemical gradient drives transport of small molecules into and out of mitochondria. ATP exported; ADP and Pi brought in. Integral membrane protein transports 1 ADP in, 1 ATP out Pyruvate exchanged for OH- Cell5e-Fig-11-14-0.jpg Fig. 11.14

Chloroplasts and Other Plastids Chloroplasts: organelles for photosynthesis: Convert CO2 plus H2O to carbohydrates Synthesize amino acids, fatty acids, and lipids of their membranes. Similar to mitochondria: generate metabolic energy, evolved by endosymbiosis, contain own genome replicate by division.

Figure 11.15 Structure of a chloroplast Chloroplasts are larger and more complex: double membrane — chloroplast envelope. internal membrane system, thylakoid membrane, network of flattened discs (thylakoids), arranged in stacks (grana) 3 internal compartments: intermembrane space stroma, ~ mitochondrial matrix thylakoid lumen Electron transport, chemiosmotic generation of ATP in thylakoid membrane, not in intermembrane space Cell5e-Fig-11-15-0.jpg Fig. 11.15

**Comparison chemiosmotic mechanism locations Fig 11.16 Chemiosmotic generation of ATP in chloroplasts and mitochondria **Comparison chemiosmotic mechanism locations Cell5e-Fig-11-16-0.jpg Fig. 11.16

Chloroplasts and Other Plastids Chloroplast genome reflects evolutionary origins from photosynthetic bacteria. Circular DNA molecules, multiple copies, Encode RNAs, proteins for gene expression, photosynthesis Rubisco catalyzes addition of CO2 to ribulose-1,5-bisphosphate during the Calvin cycle. Rubisco is critical enzyme for photosynthesis,

Chloroplasts and Other Plastids Proteins from cytosolic ribosomes imported after completion N-terminal transit peptide Guidance complex Proteolytic cleavage Toc complex Hsp70 chaperones Tic complex SPP stromal processing peptidase Fig. 11.17

Fig 11.18 Import of proteins into the thylakoid lumen or membrane Thylakoid proteins have second signal sequence, (exposed after cleavage of transit peptide). 3 paths: Chaperones + charge SRP (signal recognition particle) Cell5e-Fig-11-18-0.jpg Fig. 11.18

Chloroplasts and Other Plastids of Plants Double-membrane organelles including chloroplasts Plastids contain same genome, differ in structure and function. Chloroplasts unique: internal thylakoid membrane and photosynthesis Classified by pigments

Fig 11.19 Electron micrographs of chromoplasts and amyloplasts Chloroplasts contain chlorophyll. Chromoplasts contain carotenoids: result in yellow, orange, red colors of flowers and fruits Leucoplasts are nonpigmented - store energy sources in nonphotosynthetic tissues. Amyloplasts store starch Elaioplasts store lipids Cell5e-Fig-11-19-0.jpg

Chloroplasts and Other Plastids Plastids develop from proplastids, small undifferentiated organelles Mature plastids change. Chromoplasts from chloroplasts, in ripening fruit. Proplastids arrested at intermediate stage (etioplasts). In light, etioplasts develop into chloroplasts. Fig. 11.20

4. Photosynthesis: ultimate source of energy for biological systems: Light reactions: energy from sunlight drives synthesis of ATP and NADPH, coupled to formation of O2 from H2O. Dark reactions: ATP and NADPH drive glucose synthesis CO2 plus H2O form sugars

Fig 11.22 Organization of a photocenter Sunlight absorbed by photosynthetic pigments - chlorophylls. Photocenters in thylakoid membrane have pigment molecules Absorption of light excites electron, converts light energy to potential chemical energy. Electrons transferred through membrane carrier chain, results in synthesis of ATP and NADPH Cell5e-Fig-11-22-0.jpg Fig. 11.22

Fig 11.25 Electron transport and ATP synthesis during photosynthesis Photosynthesis: electron transport chain 4 complexes on thylakoid membrane. 2 photosystems (photosystems I and II); split H2O Cytochrome bf complex NADP reductase forms NADPH H+ gradient in thylakoid lumen ATP synthase Cell5e-Fig-11-25-0.jpg

Fig 11.27 The pathway of cyclic electron flow Cyclic electron flow uses electrons from Photosystem I only, generates extra ATP but not NADPH Fig. 11.27 Cell5e-Fig-11-27-0.jpg

Summary photosynthesis: Thylakoid membrane impermeable to protons, is permeable to other ions, particularly Mg2+ and Cl– Difference more than 3 pH units between stroma and thylakoid lumen → lot of energy across membrane. Each pair of electrons gives 2 protons at photosystem II, 2–4 protons cytochrome bf complex. 4 protons for synthesis of 1 ATP: each pair electrons yields 1 to 1.5 ATP. Cyclic electron flow yields 0.5 to 1 ATP per pair electrons.

Peroxisomes Peroxisomes: Single-membrane-enclosed organelles that contain diverse metabolic enzymes (peroxins) no genome Fig. 11.28

Peroxisomes Peroxisomes break down substrates by oxidative reactions, produce hydrogen peroxide. Peroxisomes contain catalase: converts H2O2 to water or uses it to oxidize other organic compound. Peroxisomes synthesize lipids, amino acid lysine. In animal cells, cholesterol and dolichol are synthesized in peroxisomes and in ER. In liver, peroxisomes synthesize bile acids from cholesterol Fig. 11.29

Peroxisome assembly Peroxisomes Begins on rough ER: 2 peroxins localize. Pex3/Pex19-containing vesicles bud off ER PTS1,2 signals target proteins from free ribosome to join peroxisome Signals recognized by receptors and protein channels Protein import, addition of lipids results in peroxisome growth, division. Enzyme content, metabolic activities of peroxisomes can change Fig. 11.33

lethal within first 10 years of life, Peroxisomes Diseases from deficiencies in peroxisomal enzymes, or failed import into peroxisome. Zellweger syndrome, lethal within first 10 years of life, results from mutations in at least 10 different genes affecting peroxisomal protein import. Peroxisome biogenesis disorders (PBD) – part of leukodystrophies. Damage white matter of brain, affect metabolism in blood and tissues.

Review Questions: What 2 properties of mitochondrial inner membrane give it unusually high metabolic activity? What roles do molecular chaperones play in mitochondrial protein import? Compare/ contrast import of proteins into mitochondria and into chloroplast – membrane vs. cytoplasm 11. How are proteins targeted to peroxisomes?