BCHM 4720: Exam II
Q cycle (Part of complex III function) Cont.
-Bob, Mary, and Sue the story of the 3 Q "Q pool" -Freely mobile in the membrane
Explain the toxic effects of cyanide and carbon monoxide.
-Both inhibit the ability of complex IV to become oxidized. -This decreases flux through the ETC -When flux through the ETC is inhibited proton pumping is disabled and ATP cannot be produced -ATP can't be produced because complex IV is inhibited
How do C4 plants work? What are some examples of C4 plants?
-C4 plants work by transporting oxygen to a 2nd compartment to concentrate it so that rubisco doesn't bind oxygen at high temperatures. -CO2 reacts with pyruvates to form a 4 carbon molecule that can be decarboxylated to form Co2 -Some examples are: -tropical plants -grasses -sedges (grassy bush things... think crab grass) -corn
In first 3 Steps of of Calvin Cycle
-CO2 + 4e- --> C1 aldehyde of GAP -2NADPH --> 2NADP+ + 4e- + 2H+ -Oxidation of 2 NADPH drives 4e- reduction of CO2 -Reductive Pentose Phosphate Pathway -NADPH --> NADP
Question in image on the right:
-Calculating the standard free energy change of a reaction. Answer Below:
Dark reactions:
-Calvin Cycle -Light independent reactions -CO2 -NADPH-> glyceraldehyde 3-P --> glucose --> starch
Biosynthetic roles of Citric Acid Cycle
-Cataplerotic Reactions: CAC reaction intermediates depleted for anabolic synthesis (red) -Anaplerotic Reactions: CAC reaction intermediates generated from other metabolites (green) -Shown in the image to the right
Vectorial Transport in Cytochrome C Oxidase Cont.
-Channels in subunit 1 of Cyt. C oxidase -K channel (Lys 319): mutants block activity completely -D channel (Asp 91): mutants block H+ transport completely... catalysis incompletely -How does e- transfer shift conformers directionality? -Complex IV 2 activities: -Oxidase: 1/2O2 --> H2O -Proton pump
Pigments in Photosynthesis
-Chlorophyll a,b: absorb red and blue light (are green) -Use Mg -carotenoids and phycobilins: absorb blue, green light (are red)
Chloroplast vs. Mitochondria
-Chloroplast: -Weird membrane -Super fluid -galacto lipids -Lots of protein -granae: disks 5 um long -Highly impermeable inner membrane -Outer membrane is permeable -Thylakoid membrane contains the components required for the light reactions of photosynthesis (i.e.): -Light harvesting proteins -Reaction centers -Electron transport centers -ATP synthase -[H+ are pumped into the Thylakoid (Tk) space from stroma
(II) Citrate Synthase
-Citrate (Product inhibition) -Succinyl CoA (Downstream product inhibition) -NADH (Allosteric inhibitor/OA substrate availability
TCA transport proteins:
-Citrate: TCA transport system -Malate/alphaketoglutarate: Malate/alphaKG carrier (antiporter)
Complex III: Coenzyme Q-cytochrome C Oxidoreductase (Cytochrome bc1 complex)
-CoQH2 (ubiquinol) <--> CoQ (ubiquinone) +2e- + 2H+ (-0.045 V) -2 cytochrome c (Fe3+) + 2e- <--> 2 cytochrome c (Fe2+) (0.235 V) - dEo' = 0.0190 V - dGo' = -36.7 kJ/mol (can make ATP)
Two redox cofactors of ETC
-Coenzyme Q: Ubiquinone + 2e- <--> Ubiquinol -Moves in membrane -Cytochrome C: Fe3+ + e- <--> Fe2+ -"skates" on top of membrane -Heme protein -Most conserved protein on earth (very important/ functional) -Small molecules -Moveable
In your own words, explain how some of Dr. Mitchell's personality traits (as portrayed in the 'oxphos wars') made the discovery possible, or how it might have made it more difficult (only use space below).
-Coming from outside of the field without preformed knowledge -unorthodoxy -Confirmation bias that later on prevented hime from accepting other people's views
Complex I: NADH: ubiquinone (CoQ) oxidoreductase Cont. x2
-Complex I generates new structures
Cytochrome B6F Complex from M. Laminosus
-Complex III in ETC -Plastocyonin in right of diagram -Cyt. bf transfers electrons from plastiquinol (QH2) to plastocyanin (Pc) -Protons from plastiquinol are released into the Tk lumen. -Cyt. bf pumps two more protons from the stroma into the lumen (generates a protonmotive force) -Mechanism is similar to the Q cycle of complex III of ETC in cellular respiration -QH2 + 2Pc(Cu2+) --> Q + 2Pc(Cu+) + H+(Tk lumen) -Cu1+ <--> Cu2+ + 1e-
Connection between Glycolysis, TCA cycle, Electron Transport Chain, and Oxidative Phosphorylation
-Complex IV is irreversible -Number of reduced Cyt. C depends on [NADH] -[NADH] in turn regenerates the TCA cycle
Four Complexes of ETC
-Complexes I-IV: I: NADH-Q oxidoreductase II: Succinate-Q reductase III: Q-cytochrome c oxidoreductase IV: Cytochrome C oxidase -Membrane bound big protein complexes -Physically separate assemeblies -diffuse throughout the IMM -Contain multiple components that are not easily separated -Complexes pump protons
Connection between Glycolysis, TCA cycle, Electron Transport Chain, and Oxidative Phosphorylation (summary)
-Connection between the reactions are shown in the diagram on the right -1 NADH --> 10 H+ translocated -3 ATP/H+ should make 3.7 ATP/H+ -Humans have 8 Subunits in the C ring -We make 2.5 ATP/NADH
Answer:
-Conversion of glycolytic intermediates -Most of the glucose 6 phosphate enters the glycolytic pathway -the oxidative pentose phosphate pathway -All since the cell has much more ribose 5 phosphate than NADPH -Has a low concentration of NADP+ -These conditions may occur in rapidly dividing cells
Prosthetic Group
-Covalently bound cofactor
Complex IV: Cytochrome c oxidase Cont. x2
-CuB: -1 copper distorted square planar -proximal to heme a3 -peroxide between heme a3 and CuB
Complex IV: Cytochrome c oxidase Cont. x4
-Cyt C on top of the diagram -O2 reduction to H2O -partial products are dangerous (radicals) -Need 4 e- at once -Goes first to CuA because it's more exposed
Electron Chain in Photosynthetic Bacterial Reaction Center
-Cyt. C(red) starts bound (heme) -QA (quinone) is bound after step 3 -After step 5 the e- comes from QB from Cyt. bc -Cyt. bc1 is like complex III in the ETC -No oxygen produced due to cyclic e- transport -Cyt. C: water soluble -Anaerobe: an-oxygenic (no O2 production) -End Game: [H+] gradient to power F1F0 ATPase -The possible return of e- from pheophytin to the oxidized special pair is suppressed by the "hole" in the special pair being refilled by an e- from the cytochrome subunit and the e- from the pheophytin being transferred to quinone (QA) that is farther away from the special pair (steps 3 and 4). -The reduction of quinone QB on the cytoplasmic side of the membrane results in the uptake of two protons from the cytoplasm (steps 5 and 6)
Complex IV: Cytochrome c oxidase Cont. x3
-Cyt. c oxidase consists of 13 subunits -4 Cyt. C reduced -cytochrome c --> CuA/CuA --> heme a --> heme a3 --> CuB
What is the human equivalent of plastocyanin?
-Cytochrome C is the human equivalent of plastocyanin because it works to shuttle electrons (like plastocyanin)
Plant Photosystem (Complex II)
-Cytochrome b6-F complex: Cyt. b6, Cyt. F, Fe-S proteins -Mediated by plastoquinone (Q) (quinone analog)
Dimer of Mitochondrial ATP Synthase
-Dimer of 2 F1F0 ATPase complex -C E Y: rotor rotates within a b hexamer held by a b2 d
DNP
-Dinitrophenol -Active ingredient in some herbicides and pesticides -Used as a weight loss drug -Banned in 1938 by the FDA -Comeback in popularity online and sales soared in 2014 -Causes extreme overheating -People died and in 2019 people went to jail. -
C Ring rotation
-Does the C ring move unidirectionally? -Unidirectional in the CCW direction. -C ring rotates because the H+ gradient provides continuous protonation -Full rotation produces 3 ATP -Number of subunits in the C ring determines the efficiency of the machine -Human: 8 subunits -Bacteria: 13 subunits -Motors are faster with more subunits -The fewer subunits the more efficient the motor efficiency = 1/N (N = # of subunits). Each 360 degree turn produces 3 ATP so fewer subunits means less H+ needed to produce each H+
Noncyclic Electron Transport
-Driven by hv --> PSII, PSI -4e- released from H2O --> O2 -2NADP+ reduced to 2NADPH -Photophosphorylation drives ATP synthesis through establishment of H+ gradient
(I) Pyruvate Dehydrogenase Complex
-E2: Dihydrolipoyl Transacetylase -Acetyl CoA (Product inhibition) -E3: Dihydrolipoyl Dehydrogenase -NADH (Product inhibition) -Both Reactions reversible
What does the proton gradient drive?
-Electron potential (dE) -Heat production -Active transport -Flagellar rotation -ATP (~P) -NADPH Synthesis (plants)
Photophosphorylation
-Electron transport drives H+ transport -stroma --> lumen -[H+]L/[H+]S >> 1 -Plastiquinone (Qox)--> plastisemiquinone (QH.)--> plastoquinol (Hydroquinone) (QH2) -Q Cycle
Fun ETC Facts:
-Electrons do not flow through the ETC unless there is ADP available to be converted to ATP -Respiratory (acceptor) control: regulation of Oxidative Phosphorylation by ADP. -Inhibitory Factor 1 (IF1): prevents ATP hydrolysis when oxygen is not available to accept the electron of the ETC. -Non-shivering thermogenesis: -UCP-1 (conserved) dissipates proton gradient (fatty acids) in brown adipose tissue (rich in mitochondria). -Uncouples oxidative phosphorylation and results in heat production instead of ATP -Brown adipose tissue is present in hibernators (e.g. marmots/bears)
Electron Transport Chain (ETC)
-Electrons passed through protein/cofactor complexes -Complexes convert NADH or FADH2 oxidation to a gradient of H+ -[H+] IMS >> [H+] matrix -Energy from the proton gradient is used to generate ATP -This coupling is called: oxidative phosphorylation -Oxidation of 1 NADH ~= 2.5 ATP -Oxidation of 1 FADH2 ~= 1.5 ATP -Why is there slippage (i.e. nonstoichiometry) in this process? - ~30% efficient -Under physical conditions ~70% efficient
Matrix
- <50% water (very gloopy) -contains enzymes of the CAC and pyruvate dehydrogenase complex
Percentage of Oxygen generated and ATP produced
- >80% of oxygen used - >80% of ATP produced
Write out the net reaction of the PPP if the cell needs NADPH and Ribose-5-Phosphate.
- Glucose 6 phosphate + 2 NADP+ + H2O --> ribose 5 phosphate + 2NADPH + 2H+ + 2CO2
Describe how the pentose phosphate pathway (PPP) can generate glycolytic intermediates.
- PPP can synthesize glycolytic intermediates through stage 1 (generating Ru5P) -Ru5P will then undergo stage 2 (R5P to E4P) -These will both undergo carbon shuffling reactions in stage 3 -Will ultimately form glycolytic intermediates such as GAP or F6P
Proton Motive Force (PMF)term-56
- dG(ims --> mat) = -dG(mat --> ims) - dP is the measure of dG(ims --> mat) in electrochemical units -membrane thickness of 80 A -lots of equations in the image -Typically: -psi(mat) - psi(ims) = -168 mV -pH(mat) -pH(ims) = 0.75 - dG(ims --> mat) = -20.4 kJ/mol per proton -Need more than 1 H+ to produce 1 ATP (need 3H+/ATP)
The difference in pH between the internal and external side of the mitochondrial membrane is 1.4 pH units (the external side is more acidic). If the membrane potential is 0.06 V (inside negative), what is the free energy released on transporting 1 mol of protons back across the membrane (from out to in). Assume a temperature of 310 K.
- dG(in --> out) = F(psi(out) - psi(in)) - 2.3RT(pH(out) -pH(in)) -psi: membrane potential
ATP produced from complexes
- dGo' = I + III + IV = -218 kJ/mol ~ 2.5 ATP - dGo' = II + III + IV = -148 kJ/mol ~ 215 ATP
F1F0 ATP Synthase (Complex IV)
-"Lollipop" structures inner mitochondrial membrane protruding into the matrix -F0: -water insoluble transmembrane complex -Proton translocation channel -F1: -Water soluble complex -ATP synthesis (ADP + Pi <--> ATP + H2O) -Middle and peripheral stalks (anchor point)
Calvin Cycle and PPP
-"mirror image" -Many enzymes common to the calvin cycle and the non-oxidative stage of PPP -More similarities in the image
Regulation by enzyme covalent modification
-"post translational" modification -Protein phosphorylation: -catalyzed by protein kinases (Highly specific) -Removed by protein phosphatase (unspecific)
b. How many moles of ATP could it theoretically synthesize per mole of NADH, assuming the reaction is 100 % efficient?
-1 NADH supports the transport of roughly 10 H+ so only 2 ATP can be made -Less efficient than humans -Humans in theory only use the energy from the translocation of 3 H+ per ATP -Humans make ~ 3.3 ATP per NADH
Glycolysis Overall Reaction Comparison
-2 P gained -If both glycolysis and gluconeogenesis are active at the same time then its a futile reaction (no real gain) -Both must not be active at the same time
Complex IV: Cytochrome c oxidase (Cox)
-2 cytochrome c (Fe2+) <--> 2 cytochrome c (Fe3+) + 2e- (-0.235 V) -1/2O2 + 2e- + 2H+ <--> H2O (0.815 V) - dEo' = 0.580 V - dGo' = -112 kJ/mol (can make ATP) -ATP is not directly made from this [H+] gradient
Transport of PEP and OAA from Mitochondrion to Cytosol
-2 routes for OAA "swing king" -OAA must be first converted to aspartate through aspartate aminotransferase or to malate by malate dehydrogenase.
Answer
-200.7
Gluconeogenesis Pathway Cont.
-2nd half (after aldolase) is at equilibrium -lactate comes from muscle -dG in direction of gluconeogenesis -Bypasses 3 reactions --> reciprocal regulation
Percentage of glucose made through gluconeogenesis
-50% of the glucose made in the last 20 hours is made through gluconeogenesis
Efficiency of Light Reactions and Overall Reaction
-8 photons = 8 * 170 kJ/mol = 1300 kJ/mol (input) -560 kJ/mol for the overall reaction -8 photons are required to yield 2 molecules of NADPH and 3 molecules of ATP -In cyclic phophorylation: two photons yield one molecule of ATP and no NADPH
(b) How efficient is this process compared to the energy of an einstein (mole of photons) at a wavelength 680nm? Use E=hc/λ.
-98.6% efficient -Measured / theoretical
What is the ETC and oxidative phosphorylation?
-A series of oxidation reduction reactions using NADH and FADH2 as the initial electrons donors and molecular oxygen as the terminal electron acceptor -Initial electron donors: NADH and FADH2 -Terminal electron acceptor: Molecular oxygen -Movement of electrons through complexes I through IV will generate a proton gradient -->
Plant Photosystem (Complex V)
-ATP Synthase: -H+ gradient drives ADP + Pi --> ATP -Photophosphorylation -[H+]lumen >> [H+]stroma
Photophosphorylation
-ATP synthesis driven by the protonmotive force -Very similar to animals
Nucleotide transport proteins
-ATP, ADP: Adenine nucleotide translocase (antiporter) -H2PO4-, H+: Phosphate translocase (symporter)
Substrates that do not make glucose in gluconeogenesis
-Acetyl CoA -Leucine -Lysine
No Defined transport
-Acetyl CoA -NAD+/NADH -Oxaloacetate -"smuggle" them across
Diagram of Pyruvate Carboxylase Subunit Cont.
-Acetyl CoA is an allosteric regulator (does not participate in the reaction) -Activated CO2 (carbon) is on biotin --> [flexible arm] swings into CT domain to donate pyruvate (C3) to OAA (C4)
What activates Rubisco? How does this link the dark reactions to the light reactions in photosynthesis?
-Activated by: -Mg2+ -thioredoxin -modified lysine carbamate (which binds Mg2+) -pH of 8 -ATP -These link rubsico to the light reactions since as photosynthesis continues the pH and Mg2+ concentration in the stroma increases.
Covalent modification of Enzymes
-Activation -Inhibition
Methods of transport in the Mitochondria
-Active transport (shown in image) -Facilitated diffusion -Simple diffusion
Pyruvate Carboxylase Cont.
-Anaplerotic reaction (replenishes TCA intermediates) -Occurs in mitochondria -Activated by Acetyl CoA -TCA cycle is empty (Acetyl CoA has no place to go) -Needs OAA
How animals and plants generally produce energy
-Animals: O2 + CHO --> CO2 + NADH + ATP + H20 -Plants: sunlight (hv) + H2O --> O2 + ATP + NADPH NADPH -CO2-> CHO (starches, sugars, cellulose)
Light Harvesting Antennae
-Antennae: Light harvesting complexes -In Tk membrane photosynthetic reaction centers lie next to antennae
A Special Pair of Chromosomes Initiate Charge Separation (Bacteria)
-Bacterial Photosynthetic reaction center -No Mg2+ -Absorbs infrared -Absorbs at 360 nm -Electron carrying prosthetic groups begin at bacteriochlorophylls and end at a bound quinone
Diagram of Pyruvate Carboxylase Subunit
-Biotin carboxylase is a tetramer with each monomer having 2 active sights -Enzyme is only active in presence of Acetyl CoA -CT: carboxyl transferase -Acetyl CoA binding sight on PT (pyruvate carboxylase tetramerization domain)
(a) In a plant photosystem, P680 in its ground state has reduction potential +1.0 V. Excitation of P680 by light decreases the reduction potential to -0.8 V. Calculate o'ΔG in kJ/mol for this shift in reduction potential. (b) How efficient is this process compared to the energy of an einstein (mole of photons) at a wavelength 680nm? Use E=hc/λ.
-Excitation greatly facilitates p680 oxidation -Same equation as above
F0 and F1 ATPase structures
-F0 is membrane bound -C ring is in the top right of the diagram -cytoplasm at bottom of the page
F1F0 ATP Synthase (Complex IV) Cont.
-F0: C subunits arranged in "C ring" (~8-15 C subunits) -F1: a3b3 alternating heterohexamer -a subunits: regulatory ATP bound -b subunits: catalytic (empty, ADP, ATP bound) -In the diagram: -C-E-gamma subunit is the rotor and colored yellow -Red subunit is the stator and stalk -The rotation of the gamma subunit inside the a3 b3 hexamer causes the domain movements in the B subunits
Complex II: Succinate dehydrogenase
-FADH2 <--> FAD + 2e- + 2H+ (0.040 V) -CoQ (ubiquinone) + 2e- + 2H+ <--> CoQH2 (ubiquinol) (0.045 V) - dEo' = 0.085 V - dGo' = -16.4 kJ/mol (No ATP) -TCA cycle
Complex I: NADH: ubiquinone (CoQ) oxidoreductase Cont. x3
-FMN: -like FAD - e- and H+ from NADH -Fe-S cluster's e- transfer potential depends on geometry
Complex III: Coenzyme Q-cytochrome C Oxidoreductase (Cytochrome bc1 complex) Cont.
-Fate of ubiquinol generated by complex I and II -QH2 + 2Cyt C(ox) + 2H+(matrix) --> Q + 2Cyt C(red) + 4H+(intermembrane space) -prosthetic groups: -Two Heme b: cytochrome b562 (bh) cytochrome b566 (bl) -One Heme c: cytochrome c1 -One [Fe-S] cluster: Rieske iron sulfur protein ISP -Vectorial transport of 4H+/2e-
Metabolite binding inhibition
-Feedback inhibition -Direct product inhibition -Downstream product inhibition via competition, allostery, substrate availability
Ferrodoxin/NADP+ Reductase Facts
-Ferrodoxin and NADP+ reductase transfer electrons from ferrodoxin (2xe-) to form NADPH (2e-) -Formation of NADPH occurs on the stromal side of the Tk membrane --> carbohydrate synthesis -Electron flow from H2O to NADP+ is called the Z scheme of photosynthesis -Pc(Cu2+) + Fd(ox) -hv-> Pc(cu+) + Fd(red) -dG0' = 75 kJ/mol -Need light because its unfavorable -Cyclic: 2H+/e- * 1e-/hv * 1 ATP/3H+ --> 2/3 ATP/hv -Z scheme: 12H+/4e- * 1e-/2hv * 1 ATP/3H+ --> 1/2 ATP/hv -Z scheme is more efficient per photon
Inner Mitochondrial Membrane (IMM)
-Freely permeable to only: O2, CO2, and H2O -Transport proteins exist for everything else - > 15 fold larger area than OMM due to invaginations called cristae - 75% protein by mass -Contains all the enzymes of electron transport and Ox-phosin
Mode 1: Much more ribose 5 Phosphate than NADPH is required
-Full reaction written in the image - 5G6P + ATP --> 6ribose 5-phosphate + ADP + 2H+
Amino Acid transport Proteins:
-Glutamate, aspartate: Glu/Asp carrier (antiporter)
Glucose-6-Phosphate Dehydrogenase plays a key role in the protection against Reactive Oxygen Species
-Glutathione (GSH) helps prevent damage by ROS generated over the course of metabolism -Oxidized glutathione (GSSG) is converted into reduced glutathione by NADPH -NADPH generated by G6P dehydrogenase -Glucose 6 Phosphate deficiency causes drug induced hemolytic anemia
What is glutathione and why is it necessary? How is it related to PPP?
-Glutathione is antioxidant found in plants -Useful in reducing the oxidative stress on a cell -Reduces H2O2 to H2O -The NADH produced by the PPP is needed to reduce GSSG (two glutathiones joined via a disulfide bridge) to glutathione (active form) -Useful in cells that undergoe a lot of oxidative stress (e.g. red blood cells)
Photosystem I Half Reactions
-Half reactions in the image on the right -p700(red) --> p700(ox) is super unfavorable -p700(red) -hv-> p700*(red) super favorable
P680 Reduction
-Half reactions shown in the top left of the image -Mediated by the OEC a Mn protein complex bound to PSII -In the cycle 4e- go in the cycle and 4e- enter in the last step -The OEC must store 4e- from H2O and release one by one
Response of PDH complex to the energy charge
-High Energy inhibitors: NADH, Acetyl CoA, ATP -Low Energy inhibitors: ADP, pyruvate -Inhibits PDH kinase
Why metal cofactors are needed
-In biology e- and H+ move together (hydride) so we need metals (Fe2+/Fe3+) to only trade e-
Table of Gluconeogenesis Reactions
-In image on the right
Components of the mitochondrial ETC
-In image on the right -D: e- donor -A: e- acceptor
STEP 2: PEP Carboxykinase (PEPCK)
-In mitochondria but mostly in cytosol -Driven by decarboxylation -Driven by GTP hydrolysis -Occurs often in the cytosol
Which of the following stromal changes occur in response to light that regulate the calvin cycle?
-Increase in pH -Increase in the levels of Mg2+ -Increase the amounts of reduced ferredoxin
What are the biosynthetic roles of the TCA cycle? What does cataplerotic and anaplerotic mean?
-Intermediates are drawn off for biosynthesis when the energy needs of the cell are met. -Intermediates are replenished by the formation of oxaloacetate from pyruvate and other metabolites. -anaplerotic: reactions are when CAC intermediates are generated from other metabolites. -cataplerotic: reactions are when CAC intermediates are depleted for anabolicsynthesis
Cytosol
-Intermembrane space (IMS) -Contains enzymes of glycolysis and some citric acid cycle enzymes. -pyruvate and NADH must enter the mitochondria from the cytosol -pyruvate into mitochondria and Acetyl CoA out of the mitochondria
If Complex II (succinate DH) doesn't transport protons, what happens to the energy from succinate oxidation?
-It is transferred to complex 3 through CoQ
How does Coenzyme Q move between complexes?
-It moves freely through in the membrane because of its isoprene tail
Other sources of carbon for gluconeogenesis
-Lactate (from muscle) -Amino Acids -glycerol ("BB" from fat)
Phototroph
-Light energy to feed self - ~60% are bacteria (cyano) - ~2% of light is absorbed
Light and Dark Reaction comparisons
-Light reactions make NADPH and ATP -Dark reactions use NADPH to fix CO2 --> glucose -RuBisCo: CO2 fixation -glucogenesis -Pentose Phosphate Pathway
Pentose Phosphate Pathway (PPP) Summary
-List of pathways that require NADPH is in the top left of the image -Glyceraldehyde 3 phosphate is a Sciff base in the transaldolase step -Thiamine pyrophosphate TPP forms a carbanion in the transketolase step -Transaldolase: Steps listed in the left of the image -Transketolase: Steps listed in the right of the image
Observation of ATP driven rotation in ATP synthase
-Long actin arm with fluorescent label on the end attached to gamma subunit. -Counter clockwise rotation of the gamma subunit -a3b3 is tacked down peripheral arm -gamma subunit in the a3b3 center (charge neutral) -As gamma rotates it induces conformational changes in directionality L --> T --> O (loose --> tense --> open) -This drives directionality of ATP synthesis Differences in ATP concentration: -At 200 nM of ATP: -Reaction goes backward -ATP + H2O --> ADP + Pi - T --> L --> O -At 20 nM of ATP: -Occasionally goes in the reverse direction - O --> L --> T
The Electron-Transfer Potential of an Electron
-Measured as redox potential (Eo') -Measure of a molecule's tendency to donate or accept electrons -Delta Go' = -nFdeltaEo' (n is the number of electrons transferred and F is the faraday constant)
Complex IV: Cytochrome c oxidase Cont. x5
-Mechanism of Cyt. C in the image on the right -Reduction of O2 by Cyt. C oxidase makes a resonance stabilized radical -2 e- from Fe(II) --> Fe(III) -1 e- from tyrosine -1 e- from Cu(I) -replenished with 4e- from Cyt. C. -1-4 from 4 Cyt. C to restore the system
Why are there no mitochondria in red blood cells?
-Mitochondria would use up all the oxygen that red blood cells have to transport
Ribulose Bisphosphate Carboxylase Oxygenase (RuBisCo)
-Most abundant protein on the plant -5Kg/person -Catalyzes CO2 fixation (~560 kDa) -Mechanism shown in the right of the diagram -Makes enough e- to make glucose (final product is GAP)
Gluconeogenesis Pathway
-Mostly in liver, kidney -Liver only stores ~12th worth of glucose -Gluconeogenesis goes up in the pathway on the rileft of the image and glycolysis goes down in the pathway
(III) Isocitrate Dehydrogenase
-NADH (product inhibition) -ATP (allosteric inhibitor) -ADP (allosteric activator) -Ca2+ (allosteric activator)
(IV) alphaKetoglutarate Dehydrogenase
-NADH (product inhibition) -Succinyl CoA (product inhibition) -Ca2+ (allosteric activator)
Malate/a-ketoglutarate carrier and glycerophosphate shuttle Cont. Cont.
-NADH + 1/2O2 + H+ <--> NAD+ + H2O -dEo' = 1.13 V -dGo' = 218 kJ/mol (super favorable)
Complex I: NADH: ubiquinone (CoQ) oxidoreductase Cont.
-NADH + Q + 5H+(matrix) --> NAD+ + QH2 + 4H+(intermembrane space) -vectorial proton pump -Ubiquinone: -Coenzyme Q (CoQ/Q/Q10) -oxidized quinone -"greasy" -Ubisemiquinone: -Coenzyme QH. -radical -semiquinone -Ubiquinol: -Coenzyme QH2 -reduced -active form -hydroquinone -NAD+ only deals in H+ 2e- -CoQ can do 1 or 2 e- + H+ -Fe2+/Fe3+ only deals in 1 e-
Complex I: NADH: ubiquinone (CoQ) oxidoreductase
-NADH <--> NAD+ + 2e- + H+ (0.315 V) -CoQ (ubiquinone) + 2e- +2H+ <--> CoQH2 (ubiquinol) (0.045 V) - dEo' = 0.036 V - dGo' = -69.5 kJ/mol (Formally can make ATP)
What can block components of the ETC?
-NADH-Q Oxidoreductase (complex I): -rotenone (fish poison) -amytal (barbiturate) -Q-cytochrome C Oxidoreductase (Complex III): -antimycin A (fungicide) -Cytochrome C Oxidase (Complex IV): -CN- -N3- -CO
Plant Photosystem (Complex IV)
-NADP+ Reductase: - e- from p700 chla reduce NADP+ to NADPH -Mediated by ferrodoxin -FAD is a cofactor
1) NADPH is not interchangeable for NADH
-NADPH and NADH are not metabolically interchangeable (usually) -Remember NADPH is just an ADP with a nicotinamide attached -NADH: -catabolic pathways -carbon oxidation to CO2 -ATP synthesis -NADPH: -Synthesis of: FAs, AAs, nucleotides, glutathione (reducing agent... detoxify reactive oxygen species), degradation of heme. -[NAD+]/[NADH] = 10^3 (favors oxidation) -[NADP+]/[NADPH] = 0.01 (favors metabolic reduction)
What is the oxygen evolving complex? What is the importance of Mn ions?
-OEC -Water splitting complex -Water oxidizing enzyme involved in the photo-oxidation of water during the light reactions of photosynthesis -Mn holds the electrons as they are stripped from water to make O2 -Electrons are removed four at a time but can only be placed onto Mn one by one
Draw the mechanism for the Fenton reaction. How is oxygen converted into a hydroxyl radical? Include arrows.
-OH. forms covalent adducts with DNA, protein, and lipids. -OH. (hydroxyl radical): "molecular scissors" -Through the fenton reaction forming first a super oxide then H2O2. H2O2 is then split into two OH. radicals by Fe2+.
Complex II: Succinate dehydrogenase Cont. x2
-OH. forms covalent adducts with proteins, lipids, and DNA -In complex 2 FADH is the entry point of e- -Does not pump protons but instead gives them to Q to form QH2 -fenton reaction: "biological scissors"
Step 1: Pyruvate Carboxylase
-Occurs in the mitochondria -Two phases Phase 1: -Hook P onto C --> carboxy phosphate --> carboxy biotin -Biotin = vitamin H Phase 2: -carboxybiotin --> OAA
Dark Reactions (Calvin Cycle)
-Overall Calvin Cycle in the image on the right -Rubisco enters stage 1 (fixation) -In stage 2 GAPDH: Pi + NAD+ --> NADH + ATP -Learning Goals: -explain the function of the calvin cycle -Describe how light reactions and the calvin cycle are coordinated
Energetics of Gluconeogenesis
-Overall Gluconeogenesis shown in image -Enzymes of gluconeogenesis also shown -Mitochondria: ATP>>ADP -dGo' values written below -Overall 6 P are spent 1) Pc/PEPCK vs pyruvate kinase: (1 vs -23) 2) FBPt'ase vs PFK: (-16 vs -17) 3) G6Pt'ase vs hexokinase (-14 vs -21)
Complex IV: Cytochrome c oxidase (Cox) Cont.
-Oxidase: O2 is reduced -4 Cyt. C(red) + 8H+(matrix) + O2 --> 4Cyt. C(ox) + 2H2O + 4H+(intermembrane space) -vectorial transport of 4H+ -Prosthetic groups: -Two Hemes: -Heme a -Heme a3 -Two copper centers: -CuA -CuB -They form the peroxide bridge (bind to O2) -In square planar or tetrahedral form -CuA: 2 copper center: Cu(1)-Cu(1) -Loses an electron to form Cu(II)-Cu(I) or Cu(1.5)-Cu(1.5) (delocalized) -Similar to [Fe-S]
Oxidative Stage of PPP
-Oxidation of Carbon to CO2 -Reduction of NADP+ --> NADPH +H+ a) Glucose 6P dehydrogenase: -C1 aldehyde of G6P is oxidized to acid -2e- --> 2NADPH b) 6 Phosphoglucose lactonase: -H2O ring opening -C3 alcohol further oxidized to C2 ketone -2e- --> NADPH -Overall: G-6-P --> --> CO2 + Ru5p (ribulose 5 phosphate) + 4e- 4e- + 2NADP+ --> 2NADPH + 2H+
Pyruvate Dehydrogenase (PDH)
-PDH is regulated by post translational modification (PTM) -Regulated by substrates of the TCA cycle: OA, Acetyl CoA -Regulated by the products of the TCA cycle: NADH
Plant Photosystem (5 complexes)
-PSI: light -PSII: light -Cyt. bf6: complex III (ETC) -plastoquinone: mobile e- carrier -plastocyanin: mobile e- carrier -3 transmembrane protein complexes (1, 2, and 3) linked by mobile e- carriers (plastoquinol and plastocyanin) -proton translocating ATP synthase (like us) -Light driven transport of e- from H2O to NADP form NADPH -Fd: ferrodoxin -OEC: oxygen evolving complex
Segregation of Photosystem I and Photosystem II
-PSII in stacked region to keep away from PSI -If PSII and PSI were close II680 would be attenuated for I700 -In the image the distribution of stacked (grana) to the unstacked (stroma exposed) regions of the Tk membrane is shown
Photooxidation Charge Figure
-PSII: -p680 chla dimer -pheophytin a (chla, -Mg) -plastoquinones QA, QB -2Q + 2H2O -hv-> O2 + 2QH2 -p680*: excited p680 -formally transfers e- from water to photosystem 1 in ETC
Reduced Glutathione (GSH)
-Peroxides react with GSH to form alcohol and GSSG essentially terminating the ROS
Simplified Plant Photosystem
-Photon absorption by PSII and PSI powers e- flow from water to NADP+ to make NADPH -This is used to make complex CHO, [H+] gradient (to make ATP), and O2 (waste product). -Oxygen evolving complex (OEC) and water evolving complex (WOC) are the same thing -e- is knocked loose and replaced by the e- from water
Diagram comparing Photosynthesis with Oxidative Phosphorylation
-Photosynthesis: -high e- hv -replenished from H2O -Dumped onto NADP+ -Oxidative Phosphorylation: -e- from CHO --> O2 --> H2O -H+ gradient
Thioredoxin and the regulation of the Calvin Cycle
-Photosystem I generates reduced ferrodoxin and NADPH --> regulate enzymes of the Calvin Cycle -Ferroredoxin-thioredoxin reductase: activates key enzymes by reducing disulfide bonds that control their activity -Table of enzyme regulated by thioredoxin in the image on the right
Plant Photosystem (Complex III)
-Photosystem I: -Energy form LHC drives photooxidation of chla (p700) -chla+ reduced by e- transfer from Cyt. b6-F -Mediated by plastocyanin (Cu protein) -Pc: plastocyanin -Look to the image on the right for the reaction
Light Harvesting Center II (LHC II) from peas chloroplasts Cont.
-Pigments are suspended in different orientations to harvest light from many angles and many wavelengths -Protein is used to hold the pigments in place and facilitate energy transfer. -Side view of LHC II in image on the right
What is photorespiration, what causes it and why is it wasteful?
-Plants consume O2 and release CO2 -Caused by rubisco oxygenase activity -Organic carbon becomes CO2 without producing any energy rich compounds
Model of Photosystem
-Proton gradient from NADP+ reductase to power complex 5 (ATP synthase) -E = hv [680 um] [700 um]
Components of the Electron Transport Chain (ETC)
-Proton transport across the membrane In Image: -Top: The electrons donated by NADH and FADH2 are passed to electron carriers in the protein complexes -Right: Coenzyme Q -Left: Cytochrome C: carriers electrons from complex III to complex IV -Electrons flow down an energy gradient from NADH to O2 -Catalyzed by 4 protein complexes and the energy released is used to generate a proton gradient
Vectorial Transport in Cytochrome C Oxidase
-Protons bind through side chain protonations -protons move by relay (proton wire)
Gluconeogenesis Overview
-Pyruvate (lactate and glycerol), TCA intermediates, and amino acids are made into oxaloacetate (OAA) --> PEP --> glucose 1) Reversal of glycolysis: -Except: 4 steps hexokinase, phosphofructokinase, pyruvate kinase -glycolytic enzymes -far from equilibrium -Therefore independent regulation occurs 2) Conditions that favor gluconeogenesis inhibit glycolysis and vice versa -Therefore reciprocal regulation
Tricarboxylate Transporter (TC transporter)
-Pyruvate into the mitochondria Acetyl CoA out of the Mitochondria -How pyruvate gets into the mitochondria for use in the TCA cycle -How Acetyl CoA gets out of the mitochondria for fatty acid synthesis -NADPH: Needed for fatty acid synthesis (reducing power) -ATP citrate lyase makes Acetyl CoA from citrate to be used in fatty acid synthesis -Decarboxylation of BKeto acid
Glycolysis transport proteins:
-Pyruvate: pyruvate/H+ symporter -PEP (phosphorylated pyruvate): PEP transporter
Q cycle (Part of complex III function)
-Q Cycle: mech. for coupling electron transfer from QH2 to Cyt. C -The shuttling of electrons between ubiquinol and ubiquinone in the inner mitochondrial membrane as a part of Complex III's function -QH2 carries two electrons -Cyt. C carries one electron -In one cycle 4 protons are pumped out of the mitochondrial matrix and 2 additional ones are removed from the matrix -2QH2 + Q + 2Cyt. C(ox) + 2H+(matrix) --> 2Q + QH2 + Cyt. C(red) + 4H+(intermembrane space) -Q in reduced form pick up 2H+ -In first half of the cycle two electrons of a bound QH2 are transferred. -One to Cyt. C -One to bound Q in a second binding site to form the semiquinone radical ion Qe- -In the second half of the cycle a second QH2 gives up its electrons to complex III. -One to a 2nd Cyt. C -One to reduce Qe- to QH2
Q cycle (Part of complex III function) Cont. x2
-Q cycle is an electron transport cycle in complex III
Photosystem II and the Oxygen Evolving Complex (OEC)
-Q pool in top right of the graph -p680* is a strong oxidant that removes electrons from water -Occurs at the water-oxidizing complex (aka manganese center) of photosystem II -Four photons are required to generate one O2 -The four protons used to reduce the Q molecules come from the stroma -The four protons liberated from the water are released into the lumen
Regulation of Pentose Phosphate Pathway (PPP)
-Rate of the oxidative phase is determined by the level of NADP+ (most important regulatory factor) -Dehydrogenation of glucose 6 phosphate by glucose 6 phosphate dehydrogenase is the rate limiting step of the pathway
Standard Reduction Potentials of Some Biochemically Important Half-Reactions
-Reaction potentials in diagram -Strong oxidizing agent (large positive Eo') is a strong e- acceptor -Strong reducing agent (large negative Eo') is a strong e- donor
Respirasome Supercomplex
-Reactions catalyzed by respirasome super complex -Seen through cyro EM -Proton gradient from low H+ --> High H+ -
Regulation of Citric acid Cycle
-Regulated through 4 pathways -Regulated by two main ways: -Binding of metabolites -Covalent modification of enzymes
Complex III: Coenzyme Q-cytochrome C Oxidoreductase (Cytochrome bc1 complex) Cont. x2
-Rieske [Fe-S] center coordinate Fe -has 2 His and 2 Cys -Cytochrome C (Cyt. C) picks up heme bl -Coenzyme Q-cytochrome C Oxidoreductase is a homodimer with each monomer consisting of 11 distinct polypeptide chains -Q stands for quinone -Electron transfer between quinones in the membrane and Cyt. C in the IMS is mediated by the 3 major prosthetic groups
Calculate the standard free energy for translocating one mol of protons from the mitochondrial matrix to the intermembrane space, if the difference in pH across the inner mitochondrial is 0.75 (external side is more acidic), the difference in membrane potential is 100 mV (inside negative), and the temperature is 310 K.
-Same equation as the above question
PEP Carboxykinase (PEPCK) Cont.
-Separation of pyruvate carboxylase and PEPCK: -There is no direct transport of OAA -The formation of oxaloacetate by pyruvate carboxylase occurs in the mitochondria -OAA is reduced to malate and transported to the cytoplasm where it is reoxidized to OAA with the generation of cytoplasmic NADH -PEP is then synthesized from OAA by phosphoenolpyruvate kinase. -Decarboxylation often drives unfavorable reactions (i.e. TCA cycle, PPP, fatty acid synthesis)
Progressive alteration of the forms of the 3 active sites of ATP synthase
-Shown in bottom left of the image -No two subunits are every in the same conformation
Four Modes of PPP Reactions
-Shown in the image
Four modes of PPP Mechanisms
-Shown in the image -Mode 1: Needs ribose 5P (all carbons saved) -Mode 2: Ribose 5P and NADPH needed -Mode 3: Needs NADPH -remakes G6P (gluconeogenesis) -Mode 4: NADPH and ATP needed
Transketolase Mechanism
-Shown in the image -The activated glycoaldehyde in step 4 attacks the aldose substrate to form a new C-C bond
Transaldolase Mechanism
-Shown in the image along with listed steps
Three Stages of the Calvin Cycle
-Stage 1 (fixation): Fixation of carbon by the carboxylation of ribose 1,5-bisphosphate -Stage 2 (reduction): The reduction of the fixed carbon to begin the synthesis of hexose (NADPH) -Stage 3 (regeneration): The regeneration of the starting compound ribulose 1,5-bisphosphate (similar to pentose phosphate)
Complex II: Succinate dehydrogenase Cont.
-Succinate + FAD --> fumarate + FADH2 -FADH2 + Q --> FAD + QH2 -Prosthetic groups: -FAD -3 [Fe-S] clusters -Heme b560 (Fe) -Cofactor arrangement: FAD --> [2Fe-2s] --> [4Fe-4S] --> [3Fe-4S] --> Q -Role of heme: -Not on direct path of e- transfer -Protects against reactive oxygen species (ROS) which are toxic -O2 --> HO2.- --> H2O2 -H2O2 + Fe2+ (fenton reaction) --> OH- + OH.+ Fe3+
Succinate to NAD+/FAD
-Succinate --> NAD+: -0.33 V -Succinate --> FAD: -0.031 V Not favorable
What are the purposes of the pentose phosphate pathway?
-Synthesis of NADPH and ribos 5 phosphate (R5P) -Synthesis of glycolytic intermediates
Standard reduction potentials for some biochemically important half-reactions
-Table in the image on the right
Model of E. Coli F1F0 ATPase
-The H+ wells channel through the aSubunit -With each turn one H+ flows into a well and one H+ flows out of the well ahead of it. -Essentially offset channels which (one in and one out) which the wells rotate between releasing an H+ every turn. -In subunit C (C ring) aspartic acid is the H+ acceptor
Diagram of the Core of the Water-Oxidizing Complex
-The absorption of photons by the reaction center generates a tyrosine radical which then extracts electrons from manganese ions -Mn can exist in 2+, 3+, 4+, 5+ -KoK cycle is depicted in the image on the right
In photosystem II, P680 transfers an electron to pheophytin upon excitation by a photon. How is this electron replaced? Explain the challenges with this process
-The electron is replaced by the electrons resulting from the splitting of H2O into O2 from the OEC -P680 is only able to hold onto one electron whereas the splitting of water releases 4 electrons simultaneously so those electrons need to be held onto until they can be picked up by P680
Cyclic photophosphorylation diagram
-The flow of electrons through cytochrome bf pumps protons into the thylakoid lumen.
Gluconeogenesis Facts
-The generation of free glucose (final step of gluconeogenesis) only occurs in the liver -G6P is transported to the lumen of the endoplasmic reticulum -Glucose 6-phosphatase: an integral membrane on the inner surface of the ER catalyzes the the formation of glucose from G6P -In tissues that do not dephosphorylate glucose, G6P is converted into glycogen for storage -Glucokinase: special form of special kinase in the liver -Km[glucose] ~= 5 mm (~blood glucose conc.)
Structure of Photosystem I (PSI)
-The psaA and psaB subunits are shown in red and blue respectively -The "special pair" (p700) absorbs hv -Trimer of protomers (each protomer has 2 distinct subunits)
How does bacteria establish a proton gradient?
-They establish it across their cytoplasmic membrane into the periplasm
Model of E. Coli F1F0 ATPase Cont.
-Top down view of C ring and subunit a -Another view of the intermembrane half channels
Ferrodoxin/NADP+ Reductase Diagram
-Transfers two electrons and a proton to NADP+ to form NADPH in the stroma - 2e- from PSI via ferrodoxin -FAD is a cofactor
Describe how the PPP can generate glycolytic intermediates?
-Uses glucose 6 phosphate (usually produced via glycolysis) -Fructose 6 phosphate and glyceraldehyde 3 phosphate can also enter in 2 places (glycolysis intermediates) -ribulose 5 phosphate can be interconverted to many intermediates -occurs in cytoplasm of plants so intermingling of intermediates can occur -Not a top down pathway -The intermediates produced are determined by the needs of and the intermediates available to the cell -oxidative phase: -produces ribulose 5 phosphate (precursor to sugar in DNA/RNA) -non-oxidative phase -reactions are reversible so chance for intermediates to enter at each step
You have discovered a new micro-organism whose F1F0 ATPase consists of a C-ring that has 15 subunits, as opposed to the 9 in the human complex, and 12 in bacteria. How many protons does this organism need to bring back in from the outside to synthesize one ATP? b. How many moles of ATP could it theoretically synthesize per mole of NADH, assuming the reaction is 100 % efficient?
-We need 15 H+ for one full turn -One turn makes 3 ATP -5 H+ per ATP
G6P Dehydrogenase Deficiency
-X linked - ~400 million ppl -5-25% of the population in Africa, tropical Asia, Middle East, Mediterranean -Peroxides damage cell walls and Hb becomes oxidized -Jaundice: accumulation of biliverdin since not as much NADPH is produced -Protects against plasmodium falciparum (malaria)
Why does it make sense that Rubisco is regulated by both the pH and Mg ions? Where is Rubisco found in plants?
-[H+] decreases and [Mg] increase in the stroma when light reactions are taking place -It is then optimal to have rubisco activity -Found in the chloroplast -encoded by the chloroplast genome
General Information about the pentose phosphate pathway
-also called the hexose monophosphate shunt -Takes G6P and makes NADH and ribose S phosphate -NADH: has reducing power to make stuff -Ribose S-phosphate: DNA/RNA -Bypasses: PG1, PFK, and aldolase
Chemiosmotic Hypothesis (Peter Mitchell) and PMF
-chemiosmotic hypothesis: proton gradient provides free energy for ATP synthesis -PMF: free energy available from dissipation of H+ gradient -H+(matrix) --> H+(ims): chemical gradient of H+ -Electrical gradient: separation of charge when ion passes across the membrane -Takes energy to pump H+ into IMS (across the concentration gradient)
Outer Mitochondrial Membrane (OMM)
-contains porins -Free passage of molecules up to 10 kDa -Facilitated diffusion -Smooth outer-membrane of the mitochondrion
Complex I: NADH: ubiquinone (CoQ) oxidoreductase Cont. x4
-cytoplasm portion shown in the image on the right -Distances for e- to "jump" are large -Direction of e- drops down from more -dEo' to more +dEo' -Spatially organized in protein
LHC structure:
-delocalized e- -reduced pyrrole -LHC come in repeating chains -LHC II of pea plants: -7 Chlorophyll a -5 Chlorophyll b -2 carotenoids
Diagram of complexes in membrane
-diagram in image on the right
Cytochromes
-electron transferring proteins that contain a heme prosthetic group. -The heme iron cycles between Fe2+ and Fe3+ as it accepts or donates electrons
Light reactions:
-energy from light elevates e- to excited state
Light Harvesting Center II (LHC II) from pea chloroplasts
-homotrimeric protein (3 identical monomers) -High density of cofactors - Nearly 40% of the proteins non-hydrogen atoms comprise its chlorophylls and carotenoids -Top view of LHC II in image on the right
Plant Photosystem (Complex I)
-hv hits photosystem II (chla reduced) -Energy of LHC drives photooxidation of chla (p680) -chla+ reduced by oxidation of H2O --> O2 - p680: chle(red) -(minus 1 e-)-> chl(ox) -(H2O --> O2)-> chl(red) -(Much clearer in the diagram on the right)
In thylakoid membrane
-in thylakoid membrane light harvesting complexes "attenuate" (absorb most visible light) -Very high absorption coefficients
Photooxidation
-light driven electron transport -Drives: NADPH synthesis, proton gradient
Photosynthesis in the dark:
-non-photosynthetic parts of plants
Pentose Phosphate Pathway Diagram
-oxidative phase -regenerative phase -Non-oxidative synthesis of Rib 5P, GAP, F6P -All regenerative phase reactions are at equilibrium
P680 Oxidation
-p680 lacks e- gets it back from water -2H2O --> O2 + 4e- + 4H+ (OEC) -P680 half reactions shown in the image on the right
Diagram of electron flow from H2O to NADP+ in photosynthesis
-p680*: strong oxidant -p700*: strong reducing agent -diagram has the shape of a Z
Photosystem I Charge Figure
-p700: chla dimer -Ao: A special chla -A1: phylloquinone (vitamine K) -Fx, FA, FB: Fe-S clusters -Fd: Ferrodoxin -Starts at the excitement of p700 and ends at NADP+ reductase (makes NADPH)
Complex III: Coenzyme Q-cytochrome C Oxidoreductase (Cytochrome bc1 complex) Cont. x3
-porphyrin ring has ~20 dislocalized e- -cytochrome proteins (isoprene) bind to heme prosthetic groups (no isoprene)
What are the four regulatory points in the TCA cycle? How does energy levels in the cell affect the regulation?
-pyruvate dehydrogenase (PDH) complex -citrate synthase -isocitrate dehydrogenase -alphaketoglutarate dehydrogenase -When energy is high TCA cycle is inhibited -When energy is low citric acid cycle is activated
Cyclic Electron Transport
-red light -Driven only by hv --> PSI -Electrons from PSI cycle back to Cyt. b6-F complex and plastoquinone -Drives H+ transport, ATP synthesis -No NADPH synthesis (if NADPH/NADP+ ratio is high) -No O2 synthesis (like anoxygenic bacteria)
Chemiosmotic Hypothesis
-the discovery that ATP synthesis is powered by a proton gradient is one of the two major biological advancements of the 20th (other is structure of DNA) -Theory was made by Peter Mitchell -Very eccentric and somewhat controversial -Made much of his research at a restored manor house where his research was funded in part by a herd of dairy cows
6 Stages of Gluconeogenesis
1) Pyruvate --> PEP (bypasses pyruvate kinase) 2) PEP --> Fructose 1,6 biphosphate 3) Fructose 1,6 biphosphate --> Fructose 6-phosphate HPO42- (bypasses Phosphofructokinase) 4) Fructose 6-Phosphate --> Glucose 6-Phosphate 5) Glucose 6-Phosphate --> Glucose + Pi (bypasses hexokinase)
Four Different modes of operation of the Pentose Phosphate Pathway
1) Ribose 5 Phosphate needs exceed the need for NADPH 2) The NADPH and ribose 5 phosphate needs are balanced 3) More NADH is needed than ribose 5 phosphate 4)NADPH and ATP are both required -Modes 1 and 2 are utilized by cancer cells -PPP is required for rapidly dividing cells -Rapidly dividing cells need ribose 5 phosphate for nucleic acid synthesis and NADPH for fatty acid and membrane synthesis (e.g. cancer cells) -Glyolytic intermediates are diverted to the PPP by the expression of pyruvate kinase isozyme (PKM), which has low catalytic activity
How does H+ translocation drive ATP synthesis
1. Translocation of H+ from IMS ("out") to matrix ("in") 2. Catalysis of ADP + Pi --> ATP + H2O 3. Coupling of H+ translocation to ATP synthesis -Asymmetry of F1 a3b3 subunits -b subunit forms 3 different conformers -ATP bound (L) ("lose") -ATP bound (T) ("tense") -Empty (O) ("open")
What are the three steps of the calvin cycle?
1. carbon fixation 2. reduction 3. regeneration (of rubisco the CO2 acceptor)
Order the components of the ETC to outline the flow of electrons from NADH to O2 (put correct numbers).____Q-cytochrome c oxidoreductase ___ubiquinone ___cytochrome c ____NADH-Q oxidoreductase ___Cyt.c oxidase
1: NADH-Q oxidoreductase 2: ubiquinone 3: Q-cytochrome c oxidoreductase 4: cytochrome c 5: Cytochrome c oxidase
Mode 4: Both NADPH and ATP are required
3G6P + 6NADP+ + 5NAD+ + 5Pi + 8ADP --> 5pyruvate + 3CO2 + 6NADPH + 5NADH + 8ATP + 2H2O + 8H+
Question in image on the right:
Answer below:
Four Modes of PPP:
Below
Recitation Worksheet Questions:
Below
Complex I:
Below:
Complexes I-IV of ETC
Below:
Light Reactions:
Below:
Mitochondrial Transport Proteins
Below:
Photosynthesis in the light:
Below:
Cool Stuff on the Mitochondria
Below: (Subsection of ETC and oxidative phosphorylation)
ETC and Oxidative Phosphorylation: Overall Reaction
C6H12O6 (glucose) --> --> --> 6Co2 + 24e- (stored in 10NADH and 2FADH2) 6Co2 + 24e- + 24H+ --> 12H2O -O2 consumed in mitochondria -How are the electrons from NADH and FADH2 transferred to O2? -How does this drive ATP synthesis?
CHO catabolism (respiration)
CHO --> CO2 + ATP + NADH + NADPH -glycolysis, oxphosin, pentose phosphate -Just like in humans
What are the differences between cyclic and non-cyclic electron transport in photosynthesis
Cyclic: -leads to the formation of ATP and NADPH -e- go from H2O --> PSII --> PSI --> --> -->NADPH Noncyclic: -Only some ATP is produced -e- go from PSII --> PSI and back again
Mode 3: Much more NADPH than ribose 5-phosphate is required
G6P + 12NADP+ + 7H2O --> 6CO2 + 12NADPH + 12H+ + Pi
Mode 2: The needs for NADPH and Ribose 5-phosphate are balanced
G6P + 2NADP+ + H2O --> ribose 5-phosphate + 2NADPH + 2H+ + CO2
Malate/a-ketoglutarate carrier and glycerophosphate shuttle Cont.
Glycerophosphate Shuttle: -Brain/skeletal muscle -less efficient -Not a transporter -Irreversible: can go across concentration gradient -glycerophosphate shuttle shown in image on the right
Where does the ETC and oxidative phosphorylation occur?
Inner Mitochondrial membrane
Energy source for the liver
Makes glucose from non-CHO source
Malate/a-ketoglutarate carrier and glycerophosphate shuttle
Malate-Aspartate shuttle: -transports e- across membrane (IMM) -Malate transportation is fully reversible and goes with the concentration gradient -malate dehydrogenase: swapping of utility groups between AA and ketoacid -H+ goes with aspartate into the cytosol depleting the H+ gradient -Malate-aspartate shuttle in image on the right -electrons of cytosolic of NADH are transported to mitochondrial NAD+
G6P dehydrogenase deficiency Cont.
Most common human enzymopathy. Mutant enzyme breaks down quickly. Body responds to deficiency by producing more enzyme in new RBCs. When oxidative stress increases, anemia can occur.
What are the four ways that light energy can be transferred or dissipated? Name an example of each in the process of light energy being absorbed by LHCs. Draw a Jablonski diagram representing these forms of energy dissipation.
NEED TO COME BACK AND COMPLETE THIS -Fluorescence -Internal conversion -Excitation transfer -photo oxidation NEED JABLONSKI DIAGRAM AND NAME OF EACH PROCESS
Auxotroph
Needs to consume CHO
Photorespiration (O2 --> CO2)
O2 --> CO2 -Alternate dark reaction pathway -Occurs at high [O2], low CO2, High T -dissipates energy
Oxphosin
Oxidative phosphorylation
E1: Pyruvate Dehydrogenase
PDH Kinase: Trigonal bipyramidal intermediate -E1-Ser (active) --> E1-phospho-Ser (inactive) PDH Phosphatase: -E1-phospho-Ser (E1-pSer) (inactive) --> E1-Ser (active) -Both reactions favorable in the direction written
Pentose Phosphate Pathway vs Calvin Cycle
Pentose Phosphate Pathway: -Decarboxylate and oxidize C6 --> generate NADPH -Generates a series of 3, 4, 5, 6, 7 carbon sugars (ribose 5 Phosphate) Calvin Cycle: -Carboxylate (carbon fixation) C5, cleave, reduce to 2xC3 with NADPH --> sucrose, starch -Generates a series of 3, 4, 5, 6, 7 carbon sugars (ribose 5 phosphate, regenerate ribulose 1,5 bisphosphate) Shared between the two pathways: -transketolases -transaldolases
How does ETC and oxidative phosphorylation occur?
Through the oxidation of NADH --> Membrane potential --> ATP synthesis
Red blood cells and Nerve Cells
Use only glucose
Active transport of H+
a) (Q cycle) e- transport via plastoquinone (8H+/4e-) b) (OEC) 2H2O --> O2 + 4H+ (4H+/4e-) -total of 12H+/4e- -Protonmotive force (dP): Driving force for ATP synthesis -Free energy required to transfer H+ from the stroma to the lumen -Chemical gradient -Electrical gradient -pH(lumen) < pH(stroma) -dp = pH(lumen) - pH(stroma) < 0 (3-4 pH units) -(in mitochondria 0.7) -Equation for dG and values in diagram on the right -Protonmotive force: free energy available from dissipation of proton gradient -dpsi ~= 100 mV -(170 mV in mitochondria) -Counter flow of Mg2+ out of the lumen dissipates psi -Artificially high [H+] in lumen
Photooxidation
a) 2H2O --> O2 + 4e- + 4H+ (E0' = -0.82V) b) 2H+ + 4e- + 2NADP+ --> 2NADPH (E0' = -0.32V) -Overall: 2H2O + 2NADP+ --> O2 +2NADPH + 2H+ (EO' = -1.1 V) -dG0' = -nFdE0' -F = faraday constant 96 kJ/ V*mol -n = number of electrons -dG0' = 440 kJ/mol (for the reaction above...needs to be powered by sunlight)
Why the ETC and oxidative phosphorylation?
to produce ATP
Coenzyme Q
ubiquinone