Bioenergentics & Metabolism 6 BS (MicroBiology) 1st Sem

1. Bioenergentics & Metabolism
Mitochondria
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2. Mitochondria Structure & Function
Generation of metabolic energy is a major activity of all cells
Two cytoplasmic organelles are specifically devoted to energy metabolism and production of ATP
•Mitochondria
Generates useful energy derived from breakdown of lipids & carbohydrates
•Chloroplast
Use energy captured from sunlight to generate ATP and the reducing power needed to synthesize carbohydrates from CO2 and H2O
Mitochondria Structure & Function
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3. Mitochondria
Mitochondria are
◦Responsible for most of the useful energy derived from breakdown of carbohydrates and fattyacids
Converted to ATP by oxidative phosphorylation
Mitochondria are unique among cytosolic organelles in that
They contain their own DNA which encodes
tRNA, rRNA, and some other mitochondrial proteins
Assembly of mitochondria contain
•Proteins encoded by their own genome
•Proteins encoded by nuclear genome and imported from cytosol
Mitochondria Structure & Function
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4. Structure of Mitochondria
Mitochondria are surrounded by a double membrane system
•Consist of inner and outer membrane separated by intermembrane space
•Innermembrane form numerous folds (cristae) which extends into matrix
•Large surface area
•Matrix contains mitochondrial genetic system & enzymes responsible for oxidative metabolism
•Houses the machinery for aerobic respiration
Mitochondria Structure & Function
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5. Mitochondria consist of two aqueous compartments
Interior – the matrix
Between the inner and outer membranes – intermembrane space
Inner Membrane
High percentage of proteins(> 70%)
Involved in oxidative phosphorylation
•Transport of metabolites (pyruvate, fatty acids)
Impermeable to most ions and small molecules
 a property critical to determining proton gradient that drives oxidative phosphorylation
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Mitochondria Structure & Function

6. Outer Membrane
Porins – integral proteins
Large internal channel
 Allows free diffusion of molecules smaller than about 1000 daltons
Composition of intermembrane is therefore similar to cytosol with respect to ions and small molecules
Mitochondria Structure & Function
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7. Mitochondria are positioned near to locations of high energy use i-e synapses in nerve cells, muscle cells
Continuously fusing and dividing , remodel the network of mitochondria in cell, and affect function and morphology
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Mitochondria Structure & Function

8. Endosymbiotic Origin
Mitochondria contain their own genetic system
Mitochondria are thought to have evolved from bacteria that developed a symbiotic relationship in which they lived within larger cells (Endosymbiosis)
Genomes of living organisms that are similar to mitochondria are that of
α-proteobacterium Rickettsia prowazeki
It is able to reproduce only in eukaryotic cells
But unlike mitochondria it transcribes most of its own genes
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Mitochondria Structure & Function

9. Mitochondrial Genome
Mitochondrial genome are usually circular DNA molecules, like bacteria
Present in multiple copies per organelle
Vary considerably in size between different species
Genomes of human and most other animal mitochondria are only about 16kb
Larger mitochondrial genome are found in yeast (approx 80kb) and plants (more than 200kb)
e.g
Mitochondrial DNA in Arabidopsis is 367 kb, encodes only 31 proteins
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Mitochondria Structure & Function

10. Contd…
Smallest mitochondrial genome
◦Protist: plasmodium falciparum 6kb, codes for only 3 proteins
Largest mitochondrial genome
◦Protozoan Reclinomonas americana
◦69 kb and contain 67 genes
Most present day mitochondrial genome encode
◦ Small number of proteins
Mammalian mitochondria (1000 to 5000 different proteins) representing approx 5% of proteins encoded by mammalian genome
◦tRNA, rRNA
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Mitochondria Structure & Function

11. Human mitochondrial 16 Kb genome encodes
◦Circular DNA molecule
◦Maternal inheritance
Map
Origin of replication and transcriptional promoter sequences (D loop)
16sRNA, 12sRNA, 22tRNA
13 proteins (essential for oxidative phosphorylation)
Electron transfer chain complexes, including I, III, IV and V
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Mitochondria Structure & Function
The Human Mitochondrial Genome

12. Mitochondrial Genetics
Mitochondria use a slightly different genetic code than in prokaryotic and eukaryotic cells
 Human mitochondria encode only 22 tRNAs ,approx 30 different tRNAs are required to translate the universal code according to wobble rule
Translational of mitochondrial mRNA is accomplished by extreme form of wobble
U in anticodon of tRNA can pair with any of four bases in third codon position of mRNA
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Mitochondria Structure & Function

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Nonstandard codon-anticodon base pairing
Base pairing at the third codon position is relaxed, allowing G to pair with U. Abnormal base pairing, allowing phenylalanyl (Phe) tRNA to recognize either UUC or UUU codons

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Contd…
Base pairing at the third codon position is relaxed, allowing inosine (I) in the anticodon to pair with U, C, or A. Abnormal base pairing, allowing alanyl (Ala) tRNA to recognize GCU, GCC, or GCA

16. Differences between the universal and Mitochondrial Genetic codes
Codon
Universal Code
Human Mitochondrial code
UGA
Stop
Trp
AGA
Arg
Stop
AGG
Arg
Stop
AUA
IIe
Met
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Mitochondria Structure & Function

17. Mutations in mtDNA
Mitochondrial DNA can be altered by mutations
Germ-line mutations in mitochondrial DNA are transmitted from mother
•Mutation in tRNA gene
Metabolic syndrome; obesity, diabetes
•Mutation in gene that encode components of electron transport chain
Leber’s hereditary optic neuropathy; blindness
•Progressive Mutations in mitochondrial DNA
Aging
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18. Mitochondrial proteins
Contain 1000 to 5000 different proteins but nearly half of them remain unidentified (~5% of mammalian encoded proteins)
Mitochondria from different tissues contains different proteins (tissue specific functions)
Genes for mitochondrial proteins are in nucleus(95% of mt proteins)
Some of these genes were transferred to mitochondria by original prokaryotic ancestor
Cytosoloic protein synthesis mt transport
All kerb cycle enzymes/ rep/trans/translation
Complex because of mt double membrane
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Mitochondria Structure & Function

19. Transport & assembly of matrix proteins
Pre-sequence, N-terminal 20- 35 a.a target proteins to matrix
Partially unfolded by Hsp70 chaperon
◦Prevent aggregation as emerge from free ribosomes
Bind to receptors on Tom protein complex(translocase of outer membrane)
Bind Tim complex(Translocase of inner membrane)
The presequence is cleaved by a matrix protease
a mitochondrial Hsp70 binds the polypeptide chain as it crosses the inner membrane, driving further protein translocation.
A mitochondrial Hsp60 then facilitates folding of the imported polypeptide within the matrix
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Mitochondria Structure & Function

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Insertion of mitochondrial membrane proteins
Proteins targeted for the mitochondrial membranes contain hydrophobic stop-transfer sequences (second sorting signals) that halt their translocation through the Tom or Tim complexes and lead to their incorporation into the outer or inner membranes, respectively.

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Sorting proteins to the intermediate space
Proteins can be targeted to the intermembrane space by several mechanisms.
Some proteins (I) are translocated through the Tom complex and released into the intermembrane space.
Other proteins (II) are transferred from the Tom complex to the Tim complex, but they contain hydrophobic stop-transfer sequences that halt translocation through the Tim complex. These stop-transfer sequences are then cleaved to release the proteins into the intermembrane space.
Still other proteins (III) are imported to the matrix. Removal of the presequence within the matrix then exposes a hydrophobic signal sequence, which targets the protein back across the inner membrane to the intermembrane space.

22. Mitochondrial Function
Oxidative catabolism of glucose and fatty acids
The matrix contains genetic system and enzymes for oxidative metabolism
Pyruvate is transported to mitochondria, where its complete oxidation to CO2 yields bulk of useable energy (ATP) obtained from glucose metabolism
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Mitochondria Structure & Function

23. Glycolysis
Universal pathway
Glucose starting material
Sequentially broken down to pyruvate
10 steps (all enzymes are cytosolic)
◦Early preparatory step uses ATP
◦Later steps produces chemical energy
Net Yield
◦2ATP(4ATP-2ATP)
◦2NADH
◦2Pyruvate
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Mitochondria Structure & Function

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25. Kerb Cycle
•In eukaryotic cell glycolysis took place in cytosol
•Pyruvate is then transported to mitochondria where it is completely oxidized
•Pyruvate undergoes oxidative decarboxylation in presence of coenzyme A (CoA-SH) forming acetyl CoA
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Mitochondria Structure & Function

26. Kerb Cycle
Acetyl CoA enters the kerb cycle or citric acid cycle
The 2-carbon acetyl group combines with oxaloacetate (4C) to yield citrate (6C)
In the remaining reactions the 2-carbons of citrate are completely oxidized to CO2 and oxaloacetate is regenerated
The citric acid cycle completes the oxidation of glucose to six carbon molecule of CO2
Yields one GTP, three NADH and one reduced flavin adenine dinucleotide (FDAH) which is another electron carrier
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Mitochondria Structure & Function

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Citric Acid Cycle

28. Electron Transport Chain
High-energy electrons from NADH and FADH are transferred through a series of carriers in the membrane
e- carriers organized in ET complex as I, II, III, IV
Low energy electrons from IV carried on O2+ 2H to form H2O
Energy from ETC is used to pump protons to intermembrane space
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Mitochondria Structure & Function

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Transport of electrons from NADH

30. Electron Transport Chain
Electrons from FADH2 are transferred through complex II
Then carried by coenzyme Q to complex III and IV
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Mitochondria Structure & Function

31. Proton gradient & Chemiosmotic coupling
Proton gradient established across the inner membrane
Chemiosmotic coupling: Energy stored in H+ gradient is coupled to ATP synthesis
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Mitochondria Structure & Function

32. Oxidative Phosphorylation
Protons can cross through the membrane only through a proton channel (complex V)
Complex V (ATP synthase) has two units, F0 and F1, linked by slender stalk
F0 spans the inner membrane and forms a channel through which the proton move
F1 catalyzes the synthesis of ATP
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Mitochondria Structure & Function

33. Oxidative Phosphorylation
Flow of electron through F0 drives the rotation of part of F1, which act as rotatory motor to drive ATP synthesis
Four protons are required to synthesize ATP synthesis
Oxidation of one NADH yield 3ATP; oxidation of FADH2 yield 2ATP
Kerbs and glycolysis: total 38 ATP per molecule of glucose.
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Mitochondria Structure & Function

34. THE END
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