Exam 4: Chapter 19
What are other electron entry points to ubiquinone?
(A) First step of B-oxidation passes electrons from the substrate (acyl-CoA) to electron transfer flavoprotein (ETF) (B) Glyceral-3-phosphate (from triacylglycerol metabolism or by the reduction of DHAP from glycolysis) is oxidized by glycerol-3-phosphate dehydrogenase (C) Pyrimidine synthesis donates electrons to Q in the respiratory chain Endpoint: Ubiquinone
What is the Malate-Aspartate Shuttle?
(A) NADH passes electrons to oxaloacetate (forming malate) (B) Malate is transported across the membrane (C) inside the mitochondria, the reverse happens, regenerating NADH and Oxaloacetate (D) NADH goes to the respiratory chain Note: 2 pools of malate dehydrogenase, mitochondrial and cytosolic The process is energetically neutral
How do you combine half-reaction E'0 values?
Add values
What does complex III do?
Carries electrons from reduced ubiquinone to cytochrome c
What does the net H+ movement look like in electron transport?
The net energy from both reactions is mostly used to pump H+ from the matrix For each electron pair: - 4 H+ are pumped out by complex I - 4 H+ are transferred out by complex III - 2 H+ are pumped out by complex IV - 10 H+ total using NADH
How is proton-motive force created?
The proteins in the electron-transport chain created the electrochemical proton gradient by one of three means: - actively transport protons across the membrane (complex I and IV) - chemically remove protons from the matrix (reduction of CoQ and reduction of oxygen, followed by the release of protons into the intermembrane space, oxidation of QH2) - The proton-motive force provides the energy required for the reaction (ADP + Pi -> ATP/-50kJ/mol under cellular conditions)
What does complex IV do?
Transfer electrons from cytochrome c to O2
How are protons transported across the membrane in complex I?
Protons are transported by proton wires - a series of amino acids that undergo protonation and deprotonation to get a net transfer of a proton from one side of a membrane to another
What is the chemiosmotic theory?
- ADP + Pi -> ATP is highly thermodynamically unfavorable How do we make it possible? - Phosphorylation of ADP is not a result of a direct reaction between ADp and some high energy phosphate carrier - Energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient - The energy released by electron transport is used to transport protons against the electrochemical gradient
How is electron transport coupled to ATP synthesis regulated?
- As described, ATP synthesis requires electron transport - But electron transport also requires ATP synthesis - CN- blocks electron transfer between cytochrome oxidase and O2 - Venturicidin and oligomycin inhibit ATP synthase - DNP is an uncoupler-allows free passage of H+ through membrane
How does oxidative phosphorylation begin?
- Begins with electron transfers - Dehydrogenases collect electrons from catabolic pathways and transfer them to universal electron carriers
How does energy flow in cellular respiration?
- Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell - Electrons from reduced fuels are transferred to cofactors NAD+ and FAD yielding reduced NADH or FADH2 - Electrons from the reduced cofactors NADH and FADH2 are passed to proteins in the respiratory chain - In oxidative phosphorylation, energy from NADH and FADH2 are used to make ATP - Oxygen is the ultimate electron acceptor
How do you regulate oxidative phosphorylation according to cellular energy needs?
- Cellular needs can be estimated using the Mass Action Ratio (ATP)/(ADP)=(Product)/(Reactant) - In resting condition: the ratio is very high (maximally phosphorylated) - Under high energy consumption ATP levels drop (the ratio drops proportionally) - High sensitivity and the rapid response even under extreme conditions
What are electron-transferring flavoprotein and what are they used for?
- ETF - collect electrons from B-oxidation of fatty acids
What is the general chemiosmotic model for ATP synthesis?
- Electron transport sets up a proton-motive force - Energy of proton-motive force drives synthesis of ATP - ATP synthesis results from coupling H+ flux to phosphorylation
What are flavin nucleotides and what are they used for?
- FAD and FMN - 1 or 2 electron carriers - Prosthetic group (permanently associated with protein)
How do you calculate free energy of electron transport?
- For negative deltaG need positive deltaE - E(acceptor)>E(donor) - electrons are transferred from lower (more neg.) to higher (more pos.) reduction potential - Free energy released is used to pump proton, storing this energy as the electrochemical gradient - Actual free energy difference depends on concentration of reactants and products
What are cristae?
- Found in the inner membrane of the mitochondrion - Convulsions that increase the surface area
How do oxidative phosphorylation and photophosphorylation utilize electron flow to produce uphill proton transport across a membrane impermeable to protons?
- Free energy of oxidation is transformed into an electrochemical potential - Building up the proton-motive force - The flow of protons across a membrane down the concentration gradient provides free energy for ATP synthesis in mitochondria and chloroplasts
What is the Glycerol-3-Phosphate Shuttle?
- In the Skeletal muscle and brain - Delivers NADH reducing equivalents through Glycerol-3-phosphate Cytosolic - Glycerol 3-phosphate dehydrogenase moves electrons from NADH to glycerol 3-phosphate Mitochondrial - Moves electrons from glycerol 3-phosphate into the respiratory chain 2 pools of the same enzyme Reduced FADH2 (not NADH) passes the electrons to complex III Note: no membrane transport systems (direct movement into ETC) Slight cost as fewer ATP make per NADH
What happens in the matrix of the mitochondrion?
- Locations of the citric acid cycle and parts of lipid and amino acid metabolism - Lower proton concentration (higher pH) Contains: - pyruvate dehydrogenase complex - citric acid cycle enzymes - fatty acid B-oxidation enzymes - amino acid oxidation enzymes - DNA, ribosomes - Many other enzymes - ATP, ADP, Pi, Mg2+, Ca2+, K+ - Many soluble metabolic intermediates
What is the binding-change model of F0F1 ATP synthase?
- Movement of Y stalk will push ATP of one B unit and fuse ADP and Pi together to form ATP on another B unit - The third B unit has ADP + Pi - E-ADP + Pi binding is a favorable reaction with a low G - E-ATP is even more favorable with a lower G - The formation of E-ATP coupled with the flow of protons through F0 is what releases the ATP
What are nicotinamide nucleotides and what are they used for?
- NAD+ and NADP+ - common redox cofactors - NAD+ and NADP+ do not cross the inner mitochondrial membrane - 2-electron carriers - two-electron transfer or hydride ion and proton transfer (:H + H+)
What are examples of universal electron carriers?
- Nicotinamide Nucleotides (NAD+, NADP+) - Flavin Nucleotides (FAD, FMN) - Electron-transferring flavoprotein (ETF)
How does photophosphorylation promote electron flow through membrane-bound carriers?
- Occurs in chloroplast - Sunlight is captured and used to drive ATP synthesis - Captures electrons by oxidation of H2O to O2 - Excited electrons are transported through photochemical reaction centers (photosystems)
How does oxidative phosphorylation promote electron flow through membrane-bound carriers?
- Occurs in the mitochondria - Catabolism of carbohydrate, lipid, amino acids converge on cellular respiration - Redox reactions provide electrons via reduction of NADH and FADH2 - Electron-transport chain (respiratory chain) in mitochondria
What happens in step 2 of the Q cycle?
- On the P side of the membrane 2 molecules QH2 are oxidized to Q, releasing 2 protons per Q molecule (4 protons in all) into the intermembrane space - In the second stage, the semiquinone radical is converted to QH2
What happens in step 1 of the Q cycle?
- On the P side of the membrane 2 molecules QH2 are oxidized to Q, releasing 2 protons per Q molecule (4 protons in all) into the intermembrane space - Q and N side is reduced to the semiquinone radical, which moves back into position to accept another electron
What is a transporter?
- One protein or protein complex that moves solute(s) - Symport, antiport or uniport
What is the summary of electron transport via complexes I->IV?
- Passing 2 electrons from NADH through the respiratory chain NADH + H+ + 1/2O2=NAD+ + H20 - The net reaction using NADH + H+ oxidation yields (deltaE'0=1.14V, and deltaG'0=-220kJ/mol) - Complex I -> Complex IV NADH + 11H+(n) + 1/2O2 -> NAD+ + 10H+(p) + H2O - Complex II -> Complex IV FADH2 + 6H+(n) + 1/2O2 -> FAD + 6H+(p) + H2O - difference in number of protons transported reflects differences in ATP synthesis
How do we couple proton translocation to ATP synthesis?
- Proton translocation causes a rotation of the F0 subunit and the central shaft, y - This causes a conformational change within all 3 aB pairs - The conformational change in one of the 3 pairs promotes condensation of ADP and Pi into ATP - Evidence for rotation comes from running the reverse reaction (ATP hydrolysis)
What happens in the outer membrane of the mitochondrion?
- Relatively porous membrane allows passage of metabolites - Free permeable to small molecules and ions
Why does H+ translocation cause rotation?
- The Asp/Glu side chains are ionizable - After the side chain is protonated it is neutral and does not mind going into the membrane (carousel rotation) - Once the H+ rides all the way around the carousel on the Asp, it sees the Arg and is electrostatically repelled, out the other half channel
Why does chemiosmotic energy coupling require membranes?
- The proton gradient needed for ATP synthesis can be stably established across a membrane that is impermeable to ions (plasma membrane in bacteria, inner membrane in mitochondria, thylakoid membrane in chloroplasts) - Membrane must contain proteins that couple that "downhill" flow of electrons in the electron-transfer chain with the "uphill" flow of protons across the membrane - Membrane must contain a protein that couples the "downhill" flow of protons to the phosphorylation of ADP
What are standard reduction potentials?
- Think of E'0 as "electron affinity" - Electrons flow spontaneously with low E'0 to carriers with high E'0 - this makes sense of the order in which electrons flow through different carriers in the respiratory chain - At each of these steps, there is opportunity to do work in the form of proton transport (imagine a battery powering a motor that pumps protons) - Electrons move from donors like NADH->Flavoproteins->Ubiquinone->Cytochromes->Molecular oxygen - During jumps from one donor to the next we can do work (proton pumping across mitochondrial membrane)
Where does cytochrome oxidase pass electrons?
- To O2 - 4 electrons are used to reduce 1 oxygen molecule into 2 water molecules - 4 protons are picked up from the matrix in this process - these 4 additional protons are passed from the matrix to the intermembrane space
What type of reaction happens in complex I?
- Two coupled reactions: (1) Transfer of two electrons from NADH to ubiquinone is accompanied by a (2) transfer of protons from the matrix (N) to the intermembrane space (P) - Experiments suggest that about 4 protons are transported per 1 NADH NADH + Q (oxidized) + 5H+n= NAD+ + QH2 (reduced) + 4H+p - Reduced coenzyme Q picks up 2 protons - Protons are transported by proton wires: a series of amino acids that undergo protonation and deprotonation to get a net transfer of a proton from one side of a membrane to another
What is the structure of F0?
- is a proton-coupled rotor (motor) - Ring of c subunits contains Na+ or proton-binding sites on Asp (or glutamate) residues (acidic residues) - two half channels - Proton enters half channel at a, binds to a residue on the ring of c subunits, the c subunit turns like a carousel, proton then exists through second half channel
What happens in the intermembrane space (IMS) of the mitochondrion?
- similar environment to cytosol - higher proton concentration (lower pH)
What is cytochrome c?
- the second mobile electron carrier - a soluble heme-containing protein in the intermembrane space - Heme iron can be either ferrous (Fe3+, oxidized) or ferric (Fe2+, reduced) - Cytochrome c carrier a single electron from the cytochrome bc1 complex (complex III) to cytochrome oxidase (complex IV)
What are the similarities between oxidative phosphorylation and photophosphorylation?
-Both promote electron flow through membrane-bound carriers - Both utilize electron flow to produce uphill proton transport across a membrane impermeable to protons - Couple proton flow to ADP phosphorylation - These 2 processes provide most of the ATP in biology
How do you regulate oxidative phosphorylation?
1. Complex oxidation of one glucose produces 30-32 ATP - Anaerobic glycolysis produces 2 ATP - Complete oxidation of one palmitoyl-CoA produces 108 ATP 2. Aerobic oxidation and transfer of electrons to O2 produces the vast majority of ATP at a rapid rate - production of ATP is regulated to fit fluctuating metabolism needs 3. Cellular respiration (02 oxidation) is limited by (ADP) - called acceptor control
What are the 4 distinct compartments of the mitochondrion?
1. Outer Membrane 2. Intermembrane Space (IMS) 3. Inner Membrane 4. Matrix
What is the Q cycle?
2 functions: 1. Move 4 H+ to the intermembrane 2. Pass 2 electrons to complex IV - Experimentally, 4 protons are transported across the membrane per 2 electrons that reach CytC - 2 of the 4 protons come from QH2, 2 from the matrix - the Q cycle provides a good model that explains how the 2 additional protons are picked up from the matrix - 2 molecules of QH2 become oxidized, releasing protons into the IMS - 1 molecule becomes re-reduced, thus a net transfer of 4 protons per reduced coenzyme Q
What is a shuttle?
A group of proteins (often transporters) that function coordinately (system) - the transporters work together to move the same molecule(s) into and out of one cellular space
What is the ATP synthase paradox?
Catalytic formation of ATP is reversible The puzzle: - deltaG'0 for the reaction approaches 0kJ/mol - deltaG'0 is expected to be -30.5kJ/mol The solution: 1. Affinity changes 2. The terminal phosphoryl oxygen associates/dissociates several times before ATP is made Affinity Change: - Reaction favors ATP synthesis - F1/F0 affinity for ATP is higher (10^-12M) than affinity for ADP (10^-5M) - The difference in Kd is equivalent to about -40kJ/mol - ATP synthesis requires 30-50kJ/mol
What does complexes I and II do?
Catalyze electron transfer to ubiquinone from two different electron donors: NADH (complex I) and succinate (complex II)
How does FAD transfer electrons in acyl-coa dehydrogenase?
Complex II
What happens in complex IV?
Cytochrome Oxidase - Mammalian cytochrome oxidase is a membrane protein with 13 subunits - Contains 2 heme groups: a and a3 - Contains copper ions: CuA (2 ions that accept electrons from Cyt c) and CuB (bonded to heme a3 forming a binuclear center that transfers 4 electrons to oxygen) - Electrons are shared in CuA cluster - When the center is reduced the ions have formal charges Cu1+ Cu1+, when oxidized Cu1.5+Cu1.5+ - Carrier from cytochrome c to O2 - Oxygen is the final electron acceptor - Reduces 1 O2 to 2 H2O using 4 H+ from the matrix and 4 electrons harvested from 2 complete Q cycles - Functional subunits: Iron (copper center), copper centers, hemes
What happens if the H+ gradient is not used for ATP synthesis?
Diet pills sold in the 1930s uncouple oxidative phosphorylation from ATP synthesis - This instead generated heat - Intended effect was weight loss - side effect was death by hyperthermia Also called uncoupling - Preview: if a proton gradient is dissipated some other way, the energy stored in the proton gradient is released as heat - Sometimes this is a good thing - Mitochondria in brown adipose tissues have proteins in the mitochondrial membrane that allows protons to return to the matrix without making ATP
Why does rotation cause ATP synthesis?
Enz-ADP+Pi -> Enz-ATP -> ATP (Enz-ATP->ATP is the step where rotational catalysis occurs) - ATP synthesis is not favorable but the enzyme binds to ATP much much tighter than ADP/Pi - ATP binding releases so much energy that it counterbalances the high cost of making the new bond - Proton gradient drives the release of ATP - Each B subunit has 3 conformations: empty, ADP bound, and ATP bound - Y subunit (stalk) associates with only one at a time - As it rotates between subunits, it forces ATP dissociation from each subunit, one by one - 9 c subunits: 3 ATP's are made per rotation of the ring - If there are 12 c-subunits, then every 4 H+ transfers form 1 ATP
What are the 2 functional domains of ATP synthase?
F0: an integral membrane protein with a proton pore - transports protons from IMS to the matrix, dissipating the proton gradient F1: a peripheral membrane protein that (in isolation) can hydrolyze ATP (an ATPase) ATP synthase can also be used to do the reverse, the F0 subunit can help the F1 ATPase work backwards and use ATP
What catalyzes the conversion of ADP + Pi -> ATP?
F1 catalyzes this conversion Hexamer arranged in 3 aB dimers Dimers can exist in 3 different conformations - Open: B empty - Loose: B binding ADP and Pi - Tight: B catalyzes ATP formation and binds the product Binding occurs on a cyclic basis
What does the structure of the F1/F0 complex look like?
F1: contains 9 subunits (5 different ones: a3, B3, y, S, e) - Alternating a and B subunits - B subunits have the catalytic site
What are the 4 complexes of ETC and their prosthetic groups?
I: NADH Dehydrogenase and FMN, Fe-S II: Succinate Dehydrogenase and FAD, Fe-S III: Ubiquinone: cytochrome c oxidoreductase and Hemes, Fe-S/ Cytochrome C and Heme IV: Cytochrome Oxidase and Hemes (Cua, Cub)
What is Ubiquinone?
Membrane-bound universal electron carrier (coenzyme Q) - fully oxidized - long non-polar tail (isoprenoid side chain) - A lipid-soluble conjugated dicarbonyl compound that readily accepts electrons - Upon accepting two electrons, it picks up two protons to give alcohol, ubiquinol - Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side - Coenzyme Q is a mobile (within the membrane-lateral diffusion) electron carrier transporting electrons from complexes I and II to complex III
What are some problems with NADH shuttles and the solution?
NADH and Oxaloacetate moved where they are needed Problem: - Some NADH and Oxaloacetate are produced in the cytosol - They are required in the mitochondrial matrix in high concentration - NADH dehydrogenase is in the mitochondrial matrix - Mitochondrial membranes are impermeable to NADH, NAD+ and Oxaloacetate Solution: - move reducing "equivalents" and carbon skeletons through mitochondrial membranes
What happens in complex I?
NADH: Ubiquinone oxidoreductase - One of the largest macromolecular assemblies in the mammalian cell - Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes - NADH binding site in the matrix side - Noncovalently bound flavin mononucleotide (FMN) accepts two electrons from NADH - Several iron-sulfur centers pass one electron at a time toward the ubiquinone binding site (Q) - Two coupled reactions: (1) Transfer of two electrons from NADH to ubiquinone is accompanied by a (2) transfer of protons from the matrix (N) to the intermembrane space (P) - Experiments suggest that about 4 protons are transported per 1 NADH - Reduced coenzyme Q picks up 2 protons - Protons are transported by proton wires: a series of amino acids that undergo protonation and deprotonation to get a net transfer of a proton from one side of a membrane to another
How do you use the chemiosmotic theory to calculate P/O ratios (ATO/electron pair)?
P/O ratio is the number of ATP (phosphate) made per oxygen atom (2e-) consumed Key Values: Protons pumped out per pair of electrons transferred - NADH (10) - Succinate (6) Number of protons required for the synthesis of 1 ATP - one rotation gives 3 ATP (8-10 subunits in ring, depending on organism) - 9H+/3ATP -> 3H+ per ATP plus the H+ used by the phosphate translocase symporter - 4 H+ makes 1 ATP Proton-based P/O ratios - NADH (2.5->10/4) - Succinate (1.5->6/4)
What happens in the inner membrane of the mitochondrion?
Relatively impermeable, with proton gradient across it - Impermeable to most small molecules and ions, including H+ Location of electron transport chain complexes - Respiratory electron carriers (Complexes I-IV) - ADP-ATP translocase - ATP synthase (F0F1) - Other membrane transporters Convolutions called cristae serve to increase the surface area
How do electrons from biological fuels move into the electron-transport chain?
SLIDE 14 - The respiratory chain: electrons move from NADH, Succinate or other donors to Flavoproteins/Iron-Sulfur Proteins to Ubiquinone (coenzyme Q) to Cytochromes (and copper centers) to molecular oxygen (final electron acceptor)
What is uncoupling oxidative phosphorylation?
Some inhibitors block the passage of electron to O2 - Block ATP synthesis Opposite is true - Blocking ATP synthesis blocks electron transfer - Oligomycin inhibits ATP synthase and blocks electron transfer to O2
What happens in complex II?
Succinate Dehydrogenase - FAD accepts 2 electrons from succinate - part of the citric acid cycle (the only membrane-bound enzyme in CAC) - Electrons are passed, 1 at a time, via iron-sulfur center to ubiquinone, which becomes reduced to QH2 - Picks up 2 protons (to Q, not a pump) - Catalyzes the oxidation of succinate to fumarate - 4 proteins + 2 different prosthetic groups - One protein has FAD (covalent) - One protein has iron-sulfur centers - Transfers electrons from succinate to ubiquinone (does not pump protons)
How are ATP-producing pathways coordinately regulated?
The ATP/ADP ratio controls - Oxidative phosphorylation - Citric acid cycle - Glycolysis - Pyruvate Oxidation Most control is feedback inhibition As ATP hydrolysis increases, so does - electron transfer - pyruvate oxidation - ATP synthesis These events increase glycolysis and (pyruvate) All increase rapidly and proportionately
How do you transport ADP and Pi into the matrix?
Transport mechanisms move ADP/Pi into the matrix and ATP out of the matrix 1. Adenine Nucleotide Translocase (an antiport) - exchanges ADP3- for ATP4- in the intermembrane space - use the charge gradient: one more negative charge is transported out than in 2. Phosphate Translocase (a symport) - moves 1 H+ and 1 H2PO4- into the matrix - the proton decrease the electrochemical gradient
What is thermogenin signalling?
UCP1 decouples proton pump from ATP production dissipating E as heat
What happens in complex III?
Ubiquinone: Cytochrome C Oxidoreductase - uses 2 electrons from ubiquinone (QH2) to reduce 2 molecules of cytochrome c - contains: iron-sulfur clusters, cytochrome bs, and cytochrome cs - the Q cycle results in 4 additional protons being transported to the IMS - 1 iron-sulfur center - 4 total hemes - functions as a switch from 2-electron carriers (NADH< FADH2, Q) to 1-electron carriers (cytochrome Cu)
What are iron-sulfur clusters?
Universal electron carrier - One electron carriers - Prosthetic groups (iron tightly bound with sulfur as a side-chain) - Iron is associated with inorganic sulfur or Cys sulfur - Rieske Iron-Sulfur proteins: iron coordinates with 2 His instead of 2 Cys
What is a cytochrome?
Universal electron carrier - one electron carriers - Heme groups (iron coordinating porphyrin ring derivatives) - Heme groups are tightly, but non-covalently, associated with proteins - Most are integral proteins of the inner mitochondrial membrane
What is semiquinone radical?
one electron transfer (plus one H+) results in radical intermediate
What is ubiquinol?
two-electron transfer (plus two H+) results in fully reduced ubiquinone
