Biochem Exam 3
Where does acyl-carnitine breakdown and formation occur?
-Occurs during fatty acid breakdown; formed in cytosol -Broken down in matrix
What does the number of ATP synthesized by oxidative phosphorylation depend on?
The number of protons that bind to the c-subunits of ATP synthase and are transported from the inter membrane space into the matrix
How does gamma subunit contribute to ATP synthesis?
The spinning of the c-subunits in the c-ring -> causes the gamma subunit to rotate. -The asymmetry of the gamma subunit affects the conformation of the beta subunits
What are the components of the ETC that can gain or lose electrons?
The complexes (containing iron and copper atoms)
Across which membrane does the H+ proton gradient form?
The inner mitochondrial membrane
How are protons moved from inter membrane space into the matrix?
By ATP synthase or proton leak
What is the subunit unit that ultimately causes ATP synthesis? How?
C-subunit rotation --> gamma subunit rotation --> changes conformation of beta subunits --> ATP synthesis + release
What is the order of the ETC? How do you know whether a molecule has been reduced?
Comp I --> CoQ --> Comp III --> Cyt C --> Comp IV -Reduced: if it is in the step right before; e.g. CoQ is reduced by Comp I -oxidation is other order
Which complex converts oxygen to water in the ETC?
Complex IV
If one molecule decreases ATP synthesis and 02 consumption and another molecule only decreases ATP synthesis (but not 02 consumption), what complexes/ overall movement must be affected?
Molecule I: ATP synthase, Complex I, and Complex II Molecule II: Proton movement from inter membrane space -> matrix
Electron transport is initiated by reduced electron carriers in mitochondria. Describe the process for the start of electron transport in mitochondria. Include the source of the electrons that are passed through the chain.
NADH donates electrons to Complex I or FADH2 donates electrons to Complex II (or Q if from fatty acid breakdown)
For each NADH and FADH2 molecule, how many protons are moved, oxygen consumed, and ate produced at theoretically maximum levels? What are the realistic ATP produced for each molecule?
NADH: 10 protons moved, 1/2 O2 consumed, 3 ATP produced in theory, 2.5 ATP produced in reality FADH2: 6 protons moved, 1/2 02 consumed, 2 ATP produced in theory, 1.5 ATP molecules produced in reality
How many protons are moved per 2 electrons of each reduced electron carrier in the ETC? What are theoretical and actual ATP products?
NADH: 10 protons per 2 electrons --> 3 ATP theoretically; 2.5 in reality FADH2: 6 protons per 2 electrons --> 2 ATP theoretically; 1.5 in reality
What is the order of beta conformations?
Open --> Loose --> Tight -Each beta subunit is in each conformation once per full rotation of c-ring
What are the types of mitochondrial membrane transport in the outer vs inner mitochondrial membrane? What kind of molecules does each pathway move?
Outer mitochondrial membrane: much more permeable; barrier between cytosol and intermembrane space; moves small hydrophilic molecules freely through channels Inner mitochondrial membrane: barrier between intermembrane space and mitochondrial matrix; restricted permeability (similar to that of plasma membrane); uses only specific transporters to move molecules
On which "side" is the c subunit of the Fo complex more likely to be protonated and deprotonated? In which positions can each Asp rotate towards if protonated vs deprotonated?
Protonated: exposed to intermembrane space (protonated on Asp acid side chain; neutral charge); can be exposed to hydrophobic environment Deprotonated: exposed to mitochondrial matrix (deprotonated + negatively charged on Asp acid side chain); can only rotate towards hydrophilic environment
What are some metabolic intermediates that are transported across the mitochondrial membrane?
Pyruvate, malate, citrate, acyl-carnitine
When oxygen levels are low, the activity of the TCA cycle and fatty acid breakdown often decreases, but glycolysis does not. Explain why this observation makes sense.
Reduced electron carriers generated must be regenerated to continue. NADH generated from glycolysis can be converted to NAD+ by fermentation instead of electron transport, but NADH and FADH2 generated in TCA and fatty acid breakdown must be converted to NAD+ and FAD by ETC.
Describe how the A, C, gamma, and beta subunits' structure and function contributes to the overall activity of ATP synthase.
-A subunit: two hydrophilic half-channels open to the matrix and inter-membrane space. Protons from the intermembrane space bind to an aspartic acid on a c-subunit. -C-subunit: deprotonated when it reaches the half-channel open to the matrix which causes the c-subunits to rotate. The c-ring moves in one direction because it is unfavorable for the deprotonated subunit to move into the hydrophobic membrane. The spinning of the c-subunits in the c-ring causes the gamma subunit to rotate. -Beta subunit: the asymmetry of the gamma subunit affects the conformation of the beta subunits which have three possible conformations: open (low affinity for ADP and ATP and Pi), loose (high affinity for ADP and Pi) and tight (high affinity for ATP). The tight state stabilizes ATP (lowers the energy) allowing synthesis.
Movement of electrons through which component(s) of the electron transport chain result in the net movement of protons across the inner mitochondrial membrane?
-Complexes I, III, and IV -Coenzyme Q + Cytochrome C
Fatty acid synthesis and fatty acid breakdown do not occur in the same cell at the same time. Briefly explain the mechanism of reciprocal regulation.
-Malonyl CoA is an intermediate in fatty acid synthesis and inhibits carnitine transferase which is important step in fatty acid breakdown. (activates synthesis + inhibits breakdown) -ACC activity regulates the production of malonyl CoA so activity of ACC inhibits fatty acid breakdown and promotes fatty acid synthesis.
What is the oxidative portion of oxidative phosphorylation, and what are its structural components, substrates, products, and purpose?
-Structural components: ETC -Substrates: Electrons from NADH + FADH2 --> O2 through ETC -Products: NAD+ or FAD + H20 -Purpose: Create H+ gradient by moving H+ from mitochondrial matrix --> intermembrane space (gradient forms across inner mitochondrial membrane)
How is the pH of the intermembrane space and matrix affected by the ETC?
-pH of intermembrane space << than matrix when ETC is active due to accumulation of protons
How does Acyl-CoA cross to the matrix?
1. Acyl-CoA + carnitine --> acyl-Carnitine + CoA (via carnitine transferase) 2. Acyl-carnitine goes through diffusion channel in outer mitochondrial membrane to the matrix 3. Acyl-carnitine --> carnitine + Acyl-CoA -Carnitine is going in both directions and can be diffused/ used in antiport carrier
What is the order of rotation + protonation/ deprotonation for the c subunit, beginning in the intermembrane space?
1. Asp on c subunit is protonated in intermembrane space half-channel 2. Protonated c subunit rotates 3. Protonated c-subunit moves into matrix half-channel 4. Asp on c subunit is deprotonated in matrix; protons enter the matrix 5. Deprotonated c subunit moves into intermembrane half-channel
What are the four major fatty acid functions?
1. Energy source 2. Synthesis of membrane phospholipids 3. Covalent modifications of proteins 4. Signaling
What are the three possible positions for the C subunit?
1. Exposed to hydrophobic environment (only protonated + neutral c subunit can be here) 2. Exposed to a subunit + intermembrane space hydrophilic half-channel (deprotonated c subunit rotates in this direction) 3. Exposed to a subunit and hydrophilic mitochondrial matrix half-channel (usually deprotonated here)
What are the four fates of pyruvate?
1. Gluconeogenesis --> glucose 2. Amino acid synthesis 3. Acetyl Co-A (through Pyruvate dehydrogenase) 4. Fermentation (glucose --> glycolysis --> lactate)
What are six fates of pyruvate? Where does each process take place in the body/cell?
1. Lactate (via fermentation); muscles during exercise in cytosol 2. TCA intermediates (pyruvate --> acetyl CoA --> TCA); in the mitochondrial matrix when 02 present 3. glycolysis/ gluconeogensis intermediates + glucose; in the cytosol of liver cells (glycolysis all cells) 4. Malonyl-CoA; cytosol 5. Acetyl-CoA (via PDH); via PDH (matrix) 6. amino acids (cytoplasm + mitochondria)
What are the three ordered conformations of the beta subunit? Why do the beta subunits enter these different conformations?
1. Open: low affinity for ADP, Pi, and ATP 2. Loose: high affinity for ADP + Pi 3. Tight: high affinity for ATP (ATP synthesis and subsequent release is favored in this conformation); lowers free energy of ATP --> enable ATP synthesis from ADP + Pi Differing conformations: due to rotation of asymmetrical gamma subunit (caused by rotation of c subunits); beta + alpha subunits do not move and are stabilized by additional subunits
Why can cells undergoing oxidative phosphorylation have the same components and rate of ATP synthesis but different rates of oxygen consumption?
1. Proton leak: ATP generated will be less than 02 consumed; two populations could have different amounts of proton leak 2. NADH/FADH2 molecules: s. If source of energy used by the cell generates a different ratio of NADH and FADH2 (like glucose and fatty acids), then the ratio of oxygen consumption and ATP synthesis will be different
What are the five major steps of fatty acid synthesis?
1. Synthesis of malonyl-CoA (ACC enzyme) 2. Transfer to acyl carrier protein 3. Condensation of acetyl-ACP + malonyl-ACP 4. Repeated addition of 2 carbons at a time (7 rounds) 5. Release of 16C fatty acid
What are the two types of proton leaks, and under what conditions do they occur?
1. Unregulated/ basal proton leak: continuous loss of proton gradient 2. Regulated/ inducible proton leak: specific use of proton gradient (e.g. activity of membrane transporters that use proton concentration gradient to move other molecules across membrane; transport of charged molecules across inner mitochondrial membrane)
What are the two types of proton leak?
1. Unregulated/ basal proton leak: continuous loss of proton gradient; mechanisms are always active 2. Regulated/ inducible proton leak: specific use of proton gradient; part of specific processes that are not always occurring; e.g. membrane transporter activity that uses proton gradient to move other molecules across the membrane or movement of charged molecules across membrane
What is the ETC chain overall reaction, and which substrates are being oxidized/ reduced?
2NADH + 02 + 2 H+ --> (2 NAD+) + 2H20 2FADH2 + 02 --> 2FAD + 2H20 -Electrons: oxidized; Oxygen: reduced -Very large, negative delta G (irreversible)
How many FADH2 are produced for every NADH from the TCA Cycle?
3 NADH: 1 FADH2 -Complex I inhibition has a more devastating effect than Complex II inhibition on ATP synthesis
What is the structure and components of the Fo subunit of ATP synthase?
A subunit: two hydrophilic half-channels; each is open to one side of inner mitochondrial membrane (matrix and intermembrane space); protons enter in the intermembrane space C Subunit: multiple form the C ring (8-14 units); each C subunit has two alpha helix Aspartic acid residues exposed to each half-channel; protons bind to C subunit --> protonated C subunit rotates --> proton unbinds + released into mitochondrial matrix (via other a subunit half-channel)
How does regulation of ACC enable reciprocal regulation of fatty acid synthesis/ breakdown?
ACC promotes FA synthesis and inhibits breakdown, which is reciprocal regulation. 1.) In FA Synthesis: ACC converts acetyl-CoA to malonyl-CoA (intermediate in fatty acid synthesis). 2.) In FA breakdown: malonyl-CoA inhibits carnitine transferase (which transports acyl-carnitine into mitochondrial matrix for fatty acid breakdown).
What are the four substrates of fatty acid synthesis? What enzymes/ factors activate FA synthesis?
ACP, ATP, Actyl-CoA, and NADPH -High ATP, low AMP, and high ACC
What four amino acids can by synthesized from pyruvate, alpha-ketoglutarate, and oxaloacetate broken down into pyruvate? What property do they share?
ALIV (Alanine, Valine, Isoleucine, & Leucine; non-polar)
Under what conditions is pyruvate likely to produce lactate/ undergo fermentation?
ATP is needed but 02 is not sufficient;
Under what conditions is pyruvate likely to produce Acetyl-CoA for the TCA?
ATP needed + O2 is present (generates reduced electron carriers for oxidative phosphorylation) or when the TCA cycle is active
What is the role of ATP synthase in the ETC?
ATP synthase moves protons from intermembrane space --> mitochondrial matrix using energy from ETC + proton gradient -produces ATP from ADP + Pi -"Complex Five" of ETC
Explain how/why the lack of oxygen affects the rate of ATP synthesis.
ATP synthesis in mitochondria is mostly from ATP synthase (some from TCA, but TCA is inhibited in absence of Oxygen because mitochondrial NAD+ can't be regenerated and NADH inhibits regulated enzymes). ATP synthase uses proton gradient generated by ETC. Protons are moved to the inter membrane when electrons move through the electron transport chain. -O2 is the final electron acceptor. -If there is no O2 to accept electrons, no electrons will move and no protons will be moved so there is no gradient for ATP synthase to use to make ATP
Which enzyme catalyzes the synthesis of malonyl-CoA in the synthesis of fatty acids?
Acetyl-CoA carboxylase (ACC)
During FA synthesis, where is the carbon chain being attached to?
Acyl-Carrier protein
What is proton leak, and what does it result in?
Definition: The movement of protons out of inter membrane space by ways that do not involve ATP synthase Results: reduced proton gradient --> uncoupling of electron transport + ATP synthase (electrons donated + O2 consumed is irrelevant at this point) -reduces the amount of ATP made for each NADH/ FADH2 oxidized by ETC
Why does the c subunit become deprotonated?
Due to the higher concentration of protons in matrix than intermembrane space
Which FA metabolism process uses Acyl-CoA vs Acyl-ACP? Where does each process take place?
FA breakdown: Acyl-CoA intermediate (matrix) FA synthesis: Acyl-ACP intermediate (cytosol)
What are the two "pools" of FADH2 and how do they enter the ETC?
FADH2 from Fatty acid breakdown: via "indirect pathway" and enter at coenzyme Q FADH2 from TCA: made from succinate dehydrogenase; enzyme is located in complex II (doesn't need to be transported! leads to efficiency of process)
True or false: the proton gradient provides the energy needed to synthesize H20 from O2.
False; The proton gradient provides the energy needed to synthesize ATP. The synthesis of water is a result of electrons moving from complex IV to O2 which is thermodynamically favorable
What metabolic pathway uses NADPH as a substrate?
Fatty acid biosynthesis
What enzyme catalyzes most of the FA synthesis reactions?
Fatty acid synthase
What enzyme catalyzes the majority of the steps of fatty acid synthesis?
Fatty acid synthase (FAS)
What are the locations and overall purpose of the Fo and F1 subunits of ATP synthase?
Fo: lipid bilayer of inner mitochondrial membrane; enables proton movement from intermembrane space --> matrix F1: Mitochondrial matrix; generates ATP
In what direction do protons move during the ETC?
From the inter membrane space --> matrix
In what way does ATP synthase move protons? How does it do this?
From the intermembrane space --> the mitochondrial matrix -Uses energy from proton gradient generated by ETC -ADP + Pi --> ATP
In what direction does the ETC move protons?
From the mitochondrial matrix --> the intermembrane space
How do you know how many rotations a gamma subunit completes?
Gamma subunit completes one full rotation per however many c subunits are there
The gamma subunit connects the Fo and F1 subunits of ATP synthase. Explain the role of the gamma subunit in the function of ATP synthase.
Gamma subunit rotates when c-ring rotates and changes the conformation of the beta subunits
What are the structural components of the F1 subunit of ATP synthase?
Gamma subunit: asymmetrical; middle of alpha and beta subunit hexamer Alpha subunits: alternate with beta subunits; usually 3 Beta subunits: alternate with alpha subunits; usually 3 in each c-ring; only one beta subunit can be in each conformation at a time
What are three molecules that can be converted to pyruvate? What cannot be?
Glucose, lactate, and glycerol (glycerol --> DHAP -->pyruvate) -Acetyl-CoA, ketones, and fatty acids can't be converted to pyruvate
What is the phosphorylated portion of oxidative phosphorylation, and what atre the structure components, substrates, products, and purpose?
Structural Components: ATP Synthase Substrates: ADP + Pi Products: ATP Purpose: use energy of H+ gradient to synthesize ATP
Explain why the actual estimated amount of ATP generated for every molecule of FADH2is lower than the theoretical maximum (under normal circumstances). Be sure to provide one example of how this occurs
H+ are lost from gradient because they: 1. Are used for some specific process like a transporter 2. Proton leak (move out of the intermembrane space) 3. Move into the matrix through an uncoupling protein
What aspects of the structure and function of the ATP synthase F1 subunit (the subunit not in the membrane) must you know to calculate the theoretical maximum number of ATP generated by FADH2? How can you maximize and minimize the amount of ATP created?
How many beta subunits are present. Maximized: Decreasing number of beta- subunits (Decreasing the number of beta subunits decreases the number of ATP that can be made with one 360 degree rotation of the gamma subunit (1 per beta subunit) Minimized by: Increasing number of beta-subunits
What aspects of the structure and function of the ATP synthase Fo subunit (the subunit in the membrane) must you know to calculate the theoretical maximum number of ATP generated by FADH2? How can you maximize and minimize the amount of ATP created?
How many c-subunits are present in the c-ring Maximized: Increasing c-subunits (Increasing the number of c-subunits increases the number protons required to move the gamma subunit 360 degrees. This means more protons would be needed to make each ATP and therefore less ATP could be made with 10 H+ from NADH) Minimized by: decreasing c-subunits
What is the result of proton leak?
The uncoupling of electron transport + ATP synthesis -Amount of electrons donated/ oxygen consumed does NOT determine the amount of ATP actually synthesized -Loss: 20-50% physiologically; usually 0.5 ATP
Why can't Acyl-CoA cross the membrane by itself during fatty acid breakdown?
There is not a transporter for it! so it needs to go through carnitine/acyl-carnitine antiport carrier
How is acetyl CoA attached to coenzyme A (in fatty acid synthesis)?
Thioester bond
Carnitine supplements are sold and claim to increase cellular levels of carnitine. Explain why increased carnitine levels might help increase muscle performance by affecting metabolism.
Transport of acyl-CoA into mitochondria during fatty acid breakdown requires transfer of the acyl from CoA to carnitine in the cytosol, transport through the carnitine transporter in the inner mitochondrial membrane, and conversion back to acyl-CoA in the mitochondria (carntitine shuttle). -Increasing the amount of carnitine available might increase the rate of transport of fatty acids into the mitochondria for fatty acid breakdown (it's the rate limiting step for fatty acid breakdown)
True or false: electron movement provides energy to move protons into the inter membrane space.
True
True or false: four electrons are needed to completely reduce an 02 molecule
True
True or false: the Asp on c-subunits move from inter membrane space half channel to matrix half-channel in protonated state.
True
True or false: the ability of complex I to oxidize NADH is reduced under low O2 levels?
True; the electrons will not be able to move through the chain and no more electrons can be donated to the electron accepting part of complex I
What are the differences in fatty acid structure for unsaturated vs saturated fatty acids? How are fatty acids stored?
Unsaturated: 18 carbons + one double bond Saturated: 16 carbons + no double bonds Stored: as triacylglycerol (glycerol + three acyl groups) -acyl group: carbonyl + hydrocarbon chain
What aspects of the structure and function of the electron transport chain must you know to calculate the theoretical maximum number of ATP generated by FADH2?
Which complexes the electrons from FADH2 travel through (III and IV) and how many protons are pumped by each complex
How is the pH of the inter membrane space changed while proton gradient is being formed?
reduced when ETC is active (higher proton concentration in intermembrane space)
What is the purpose of doubling the molecules in the ETC overall reaction?
to prevent damaging 02 species from forming
What is the role of coenzyme Q? What would you expect to happen if Coenzyme Q is inhibited?
transfers electrons from complex I and II to complex III so electrons donated by both NADH through complex I and FADH2 through Complex -If coenzyme Q is decreased: >> the rate of electron flow would be decreased because electrons must be passed to coenzyme Q for more electrons to enter the complexes. >> The rate of protons moved across the membrane by Complex I will also be affected because the proton movement is a result o felectron movement
