3.1 ATP Synthase and Chemiosmosis Theory
Compare and contrast substrate-level phosphorylation and oxidative phosphorylation.
Both processes produce ATP from ADP and Pi, but substrate-level phosphorylation occurs when enzymes remove a "high-energy" phosphate from a substrate and directly transfer it to ADP, while oxidative phosphorylation occurs when electrons move through an ETC and produce a proton-motive force that drives ATP synthase
When NADH is supplied, which complex or electron carrier of the mitochondrial ETC is responsible for A, B, and C?
A: Complex I B: Complex I C: Q
Example of "chemiosmosis"
ATP synthase powered by a H+ gradient
Chemiosmosis Hypothesis
An electrochemical proton gradient across the inner mitochondrial membrane is the high energy intermediate • ETC creates the proton gradient -- intact mitochondrial inner membrane is required • ATP synthase uses the gradient to make ATP
Chemiosmosis and proton motive force
Chemiosmosis: use of a proton motive force to drive energy-requiring processes Proton motive force (Δp = = ΔVm + ΔpH): • The electrochemical gradient of protons • ΔVm = membrane potential • ΔpH = concentration gradient of H+
ATP synthase arrangement in the mitochondria
Clustered in dimers along the crista ridge (folds of the inner mitochondria membrane) Supports a high local proton gradient
1. Oxidative phosphorylation in mitochondria is often monitored by measuring oxygen consumption. When oxidative phosphorylation is proceeding rapidly, the mitochondria will rapidly consume oxygen. If there is little oxidative phosphorylation, only small amounts of oxygen will be used. You are given a suspension of isolated mitochondria and directed to add the following compounds in the following order, from a to d. With the addition of each compound, all of the previously added compounds remain present. Predict the effect of each addition on oxygen consumption by your mitochondrial sample. (a) ADP + Pi
Decrease of [O2] for a brief period. Rate of ATP production will be increase, and more oxygen (the final electron acceptor of ETC) will be consumed. However, when the mitochondria run out of fuel, ATP synthesis will stop and there will be no effect on [O2].
1. Oxidative phosphorylation in mitochondria is often monitored by measuring oxygen consumption. When oxidative phosphorylation is proceeding rapidly, the mitochondria will rapidly consume oxygen. If there is little oxidative phosphorylation, only small amounts of oxygen will be used. You are given a suspension of isolated mitochondria and directed to add the following compounds in the following order, from a to d. With the addition of each compound, all of the previously added compounds remain present. Predict the effect of each addition on oxygen consumption by your mitochondrial sample. (a) ADP + Pi (b) Citrate (c) Oligomycin (d) Rotenone, an inhibitor of Complex I
Decrease of [O2], although at a lower rate. Rotenone inhibits Complex I, but the presence of citrate will enable electrons to enter at Complex II.
1. Oxidative phosphorylation in mitochondria is often monitored by measuring oxygen consumption. When oxidative phosphorylation is proceeding rapidly, the mitochondria will rapidly consume oxygen. If there is little oxidative phosphorylation, only small amounts of oxygen will be used. You are given a suspension of isolated mitochondria and directed to add the following compounds in the following order, from a to d. With the addition of each compound, all of the previously added compounds remain present. Predict the effect of each addition on oxygen consumption by your mitochondrial sample. (a) ADP + Pi (b) Citrate
Decrease of [O2]. Citrate is can be metabolized by the mitochondria via the TCA cycle, which will generate NADH and FADH2 that provide electrons to the mitochondrial ETC.
Discovery of ATP synthase
Lollipop-shaped "base-stalk-knob" isolated from mitochondrial membrane • Stalk and knob: hydrolyzes ATP and synthesizes ATP in vesicles • Stalk/base: transports protons; cannot process ATP • ATPase is the "coupling factor"
Fo ATP Synthase Structure
Membrane-bound, proton-transporting base component • Upon proton binding, electrostatic interactions trigger the rotation of the stalk/base (Fo) • Proton enters one side of the channel, and exists from the other side as Fo rotates
Mitochondrial ATP synthase Structure
Mitochondrial ATP synthase = Complex V = FoF1 synthase ("o" for oligomycin-ATP Synthase inhibitor) "Knob-and-stalk" structure • F1 (knob): catalytic subunits; ATP synthesis • Fo (stalk): proton channel; spans the membrane moves three protons per ATP synthesized
Explain the relationship between electron transport and oxidative phosphorylation. What does ATP synthase look like, and how does it work?
Oxidative phosphorylation is possible via a proton gradient that is established by Redox reactions in the ETC. ATP synthase consists of a membrane-associated Fo unit and a F1 unit joined by a rotor shaft. When protons flow through the Fo unit, it spins the rotor shaft within the fixed F1 unit. This spinning shaft causes structural changes in the F1 that drives the synthesis of ATP from ADP and Pi
The mitochondrial ETC & ATP synthase
Most of the ATP produced by cellular respiration is made by a flow of protons. ATP production is coupled to the ETC by oxidative phosphorylation. The potential energy stored in the proton gradient is used to spin components of the ATP synthase to produce ATP. This process is responsible for most of the ATP made by cellular respiration.
3. After glucose is fully oxidized by glycolysis, pyruvate processing, and the citric acid cycle, where is most of its energy stored?
NADH
Where Do H+ used in ATP Synthase come from
NADH -> NAD+ +2e- + H+ ETC Using redox energy to pump H+ to generate the proton gradient H+ go through the ATP Synthase to make ATP
Generating ATP by chemiosmotic coupling
Oxidative phosphorylation Oxidation of NADH/FADH2
1. Oxidative phosphorylation in mitochondria is often monitored by measuring oxygen consumption. When oxidative phosphorylation is proceeding rapidly, the mitochondria will rapidly consume oxygen. If there is little oxidative phosphorylation, only small amounts of oxygen will be used. You are given a suspension of isolated mitochondria and directed to add the following compounds in the following order, from a to d. With the addition of each compound, all of the previously added compounds remain present. Predict the effect of each addition on oxygen consumption by your mitochondrial sample. (a) ADP + Pi (b) Citrate (c) Oligomycin
Oxygen consumption stops. Oligomycin inhibits ATP synthesis, which is coupled to the activity of the ETC.
Testing the Chemiosmotic Hypothesis
Synthetic vesicle with biochemically separate systems, but linked only by a proton-motive force • Bacteriorhodopsin: light-driven proton pump Evidence for the role of the proton motive force driving ATP synthesis via ATP synthase
Proton Motive Force
The potential energy stored in the form of an electrochemical gradient, generated by the pumping of hydrogen ions across biological membranes during chemiosmosis.
Photosynthetic ETC and ATP synthesis: photophosphorylation
The process of generating ATP from ADP and phosphate by means of a proton-motive force generated by the thylakoid membrane of the chloroplast during the light reactions of photosynthesis.
F1 ATP Synthase Structure and Mechanism
The rotation of Fo is conveyed to F1 • As Fo rotates, F1 subunit conformation changes and catalyzes ATP synthesis
Cyanide (CKN−) blocks complex IV of the electron transport chain. Suggest a hypothesis for what happens to the ETC when complex IV stops working. Your hypothesis should explain why cyanide poisoning in humans is fatal.
When complex IV is blocked, electrons can no longer be transferred to oxygen, the final acceptor, and cellular respiration stops. Fermentation could keep glycolysis going, but it is unable to fuel a cell's energy needs over the long term. The low production of ATP would result in death, with those cells that lack the capacity for fermentation dying first.
During aerobic respiration, which of the following is true? Select one: a. Oxygen acts as an efficient electron donor during aerobic respiration. b. Oxygen acts as an efficient electron acceptor during aerobic respiration. c. NAD+ acts as the final electron acceptor in the electron transport chain, and is reduced to NADH. d. Glucose reacts with oxygen to yield CO2 in a reaction that produces ATP and NADH e. Glucose is catabolized to ethanol via acetyl-CoA.
b
2. What does the chemiosmotic hypothesis claim? a. ATP is generated using phosphates taken from intermediates in the electron transport chain. b. ATP is generated using a phosphate gradient produced by glycolysis and the citric acid cycle. c. ATP is generated using a proton-motive force that is produced by the electron transport chain. d. Water is generated using electrons taken from NADH and FADH2 and transported through the electron transport chain.
c
A biochemist wants to control the initial substrate-level phosphorylation that occurs in the tracheal cells of grasshoppers once glucose has crossed the plasma membrane. She has access to the following inhibitors: Rotenone - an electron transport chain inhibitor, Oligomycin - an ATP synthase inhibitor, and TLN-232, an inhibitor of glycolysis. Which inhibitor should he use to slow down initial substrate-level phosphorylation that occurs once glucose has crossed the plasma membrane? Select one: a. Rotenone b. Oligomycin c. TLN-232 d. None of these inhibitors would be effective in preventing substrate-level phosphorylation
c
ATP synthase, as it is synthesizing ATP, accomplishes which of the following? Select one: a. Phosphorylates ADP to ATP b. Creates the high concentration of H+ in the intermembrane space of mitochondria c. Drains the high concentration of H+ in the thylakoid space d. Both a and c e. Both b and c
d
Suppose you discovered that a small number of specialized cells contain a previously uncharacterized protein embedded in the inner mitochondrial membrane. You propose that it could be a new electron transport chain member that only these cells possess. You measure its redox potential and find that it is less than that of Complex IV, but greater than that of cytochrome C. Which would be a reasonable model(s) for the function of this protein in the ETC, if indeed that's what it does? Select one or more: a. it could receive electrons from Complex IV as an alternative final electron acceptor b. it could receive electrons from Complex IV and then pass them onto oxygen c. it could receive electrons from Complex III and then pass them onto cytochrome C d. it could receive electrons from Complex III and then pass them onto complex IV e. it could receive electrons from cytochrome c and then pass them onto Complex IV
d
Oxidative phosphorylation:
energy released by redox reactions is used to make ATP (The energy to produce ATP in oxidative phosphorylation comes from an established proton gradient, not phosphorylated substrates)
