1.1.1 Pre-Midterm Biochem - Mitochondrial Shuttles, Etc & Oxidative Phosphorylation

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Front 2

Outline the aspartate-malate (heart and liver) and glycerophosphate (white muscle tissue) shuttles and their significance in regeneratinbg cytoplasmic NAD+

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The inner mitochondrial membrane is impermeable to NADH. The shuttles are means of restoring cytoplasmic NAD+ from NADH2 and harnessing ATP from NADH2 derived from glycolysis. The NAD+ regenerated can be used to continue the use of the G3P dehydrogenase reaction in glycolysis.
The glycerol phosphate shuttle converts DHAP into G3P via glycerol-3-phosphate dehydrogenase on the inner-mitochondrial membrane by oxidizing NADH into NAD+ (cytosol). The G3P is then oxidized back to DHAP by reducing FAD into FADH2 (FADH2 reoxidized to FAD by ETC) in the matrix. Cytoplasmic NADH is worth 2 ATP (only complex 3 and 4 are involved in the ECT transport from the FADH2). Shuttle is prominent in white muscle tissue.
The malate aspartate shuttle converts oxaloacetate to malate by oxidizing NADH into NAD+ (cytosol). The malate transverses the mitochondrial membrane and is reoxidized into oxaloacetate in the TCA cycle. Mitochondrial NADH is worth 3 ATP (NADH generated in the mitochondria, can enter the ETC through complex 1). Shuttle is prominent in human heart and liver.

Front 3

Describe the role of the inner mitochondrial membrane in electron transport and oxidative phosphorylation

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Oxidative phosphorylation, the coupling of oxidation (with carriers of electrons and oxygen) and phosphorylation (ADP to ATP) occurs on the inner mitochondrial membrane. The inner mitochondrial membrane is rich in protein (half are involved with ETC and oxidative phosphorylation). The chain consists of an assembly of proteins and enzymes firmly embedded in the inner mitochondrial membrane. The flow of electrons is from negative to less negative.

Front 4

Outline the different types of electron carriers found in the ETC complexes and describe the path electrons follow from NADH and FADH2 to oxygen, through the ETC complexes I through IV.

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Complex 1: NADH-Coenzyme Q Reductase (NADH Dehydrogenase)
Binds NADH (matrix) → FMN, Fe-S → CoQ (intermembrane space)
Complex 2: Succinate-Coenzyme Q Reductase
FADH2 (from TCA) → FeS → CoQ (not a transmembrane complex, cant pump protons out)
Complex 3: CoQ Cytochome c Reductase
CoQ (complex 1 and 2) → 2 cyt c (Fe2+ from Fe3+)
Complex 4: Cytochome c Oxidase
Cyt c → cyt a → Cu (bimetallic, prevents formation of superoxide anions)
cyta3 → O2
Four electrons must be supplied to the molecule of O2 simultaneously to avoid production of toxic species such as superoxide radicals.

Front 5

Discuss the mode of action and the effects of specific inhibitors of the ETC (including antimycin A, azide, hydrogen sulphide, cyanide, rotenone, piericidin A, and carbon monoxide.

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Inhibitors of complex 1 include rotenone, amytal, and piericidin A (RAP). Piericidin competes with CoQ for accepting electrons from complex 1. Inhibitors of complex 3 include actimycin A which binds to a Fe-S protein. Inhibitors of Complex 4 include N3, H2S, CN (bind ferric, 3+) form and CO (bind ferrous, 2+) form of copper.

Front 6

Explain how the electrochemical gradient is generated during electron transport.

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The production of ATP by oxidative phosphorylation in mitochondria is an endergonic process which is separate from the exergonic process of electron transport (the two are tightly coupled). The free E released from the downhill flow of electrons is coupled to the pumping of protein across the proton impermeable membrane (matrix  intermembrane space). 4H+ from complex 1; 4+ from complex 3; 2H+ from complex 4. 10 protons are pumped from NADH while 6 protons are pumped from FADH2. The proton pump thus t in the matrix (higher pH). This gradient is the potential E used in the synthesis of ATP.

Front 7

Describe ATP synthesis with reference to Mitchell's Chemiosmotic Theory.

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The chemiosmotic mechanism suggests that the chemical reaction and a transport process take place simultaneously. There are assumptions for the chemiosmotic mechanism.
1. Mitochondrial inner membrane is impermeable to protons
2. Protons can only cross the inner membrane via carriers of the ETC and ATP synthase
3. Carriers of the ETC are vectorally arranged so protons move from matrix to intrermem space
4. ATP synthase is also vectorally arranged
ATP synthase is a complex transmembrane protein (complex 5) that is separate from the ETC. It serves as a coupling agent that links the oxidation ( transport) with phosphorylation of ADP. Fo (stalk) is embedded in the inner membrane (allows H+ expelled from the mitochondrion by the pump to re-enter the matrix). F1 (knowb) projects in the matrix and is the catalytic site involved in the synthesis of ATP. As protons move through the stalk it causes rotation of the ATP synthase, molecular motor.

Front 8

Describe the mode of action of the mitochondrial ATP synthase and its inhibition of oligomycin.

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There are three different F1 conformations:
L conformation: permits binding of ADP and P
T conformation: catalytically active in bringing ADP with P to condense and produce ATP
O conformation: binding site opens freeing ATP and leaving site open to accept ADP and P
Oligomycin and DCCD (dicyclohexylcarbodiimide) bind to the Fo of ATP synthase and prevent the flow of protons into the matrix through the proton channel. The consequence is the cessation of ATP synthesis.

Front 9

Discuss respiratory control of the ETC and the P/O ratio

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Electron transport rate is tightly controlled and dependent on ADP and O2 consumption. Availability of ADP is referred to as respiratory/acceptor control of respiration. When the ATP/ADP ratio is high, the rate of O2 uptake will be low (little need to form ATP). When the ATP/ADP ratio is low, the rate of O2 uptake is high. If there is no ETC, there is ATP synthase. P/O ratio: 10 protons are pumped out for NADH and 6 for FADH2. The flow of 4 protons back into the matrix is needed to synthesize ATP including one to use the translocase. Thus the P/O ratios are 2.5 and 1.5.

Front 10

Define uncoupling of oxidative phosphorylation with reference to the different types of uncouplers (including 2,4-DNP, valinomycin, gramicidin A).

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Uncoupling is the state where electron transport occurs without ATP synthesis and results from the inability to maintain a proton gradient. Uncouplers disrupt the tight coupling between electron transport and ATP formation so that the free E from electron transport is not conserved in the formation of ATP. There is no oxidation without phosphorylation. The free energy of electron transport is released as heat and there is loss of respiratory control The brake on electron transport by ATP formation is removed and there is accelerated respiration rate without ATP formation. 2,4 dinitrophenol (DNP) is a lipophilic weak acid that moves protons from the intermembrane space back into the matrix. Valinomycin is a transporter of K+ ions and is lipid soluble. It nullifies the potential gradient of the proton motive force but the pH gradient is still present. Gramicidin A forms a H-phobic transmembrane channel through which ions (mainly K+) can cross the membrane and deplete the ionic gradient and the membrane potential.

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Discuss transport of ADP into, and ATP out of the mitochondrion and inhibitors of this process (including atractyloside and bongkrekic acid)

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ATP is produced in the matrix and needs to be actively transported out into the inter membrane space. The ATP/ADP translocase is an integral membrane protein that binds ADP on the outside of the inner membrane ATP on the matrix and moves ATP out and ADP in. Atracyloside binds to the translocase side facing the cytosol, while Bongkrekic acid binds to the translocase side facing the mitochondrial matrix. Oxidative phosphorylation stops soon after either inhibitor is added showing that the ATP translocase is essential.

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Differentiate between: direct inhibitors of the ETC, uncouplers of oxidative phosphorylation, ATP synthase inhibitors (oligomycin) and inhibitors of the ATP/ADP translocase.

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Front 13

List diseases involving mutations in mitochondrial DNA: Leber Hereditary Optic Neuropathy, Myoclonic Epilepsy and Ragged-Red Fiber Disease, MELAS, Aminoglycoside Induced Deafness

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Defects in oxidative phosphorylation are often due to alterations in mitochondrial DNA which has a much higher mutation rate than nuclear DNA and is maternally inherited.
Leber’s Hereditary Optic Neuropathy: Point mutation in a gene for NADH dehydrogenase and for cytochome b. Rare, late onset, affects CNS optic nerves and causes bilateral loss of vision in early adulthood.
Myoclonic Epilepsy/Ragged-Red Fiber Disease: Mutation in the gene for the tRNA for lysine. Symptoms include uncontrollable muscle jerks. Skeletal muscle fibers contain mitochondria with unusual shapes (ragged red fibers). In most cases, mutation produces multiple deficiencies in the enzyme complexes of the ETC.
MELAs: (mitochondrial myopathy, encephalopathy, lactacidosis, stroke). Mutation in the gene for the tRNA for leucine. Progressive neurogenerative disorder that presents between 5-15yrs. Associated symptoms are progressive neurodegeneration and stroke-like episodes.
Aminoglycoside Induced Deafness: A heredity susceptibility to aminoglycoside antibiotics from a mutation in mitochondrial DNA. Aminoglycosidases in levels at the therapeutic range can cause irreversible hearing loss. Hypersensetivity to aminoglycosidsaes to mitochondrial ribosomes and hence loss of ATP making machinery. Cochlea have large E demands and can be profoundly affected by these disorders.