BIOL2056 Cell Biology Lecture Notes PDF

BIOL2056: Cell Biology Bioenergetics Dr Philip T.F. Williamson [email protected] Resume of Lecture 1 Key Points Origins of mitochondria Structure of mitochondria Role of the compartments/membranes Location and transport within the cell Lectures 2 and 3 How do mitochondria conve...

BIOL2056: Cell Biology Bioenergetics Dr Philip T.F. Williamson [email protected] Resume of Lecture 1 Key Points Origins of mitochondria Structure of mitochondria Role of the compartments/membranes Location and transport within the cell Lectures 2 and 3 How do mitochondria convert NADH into ATP Bioenergetic and the chemiosmotic theory Energy Conversion: 2H2 + O2 Direct Combustion Biological Oxidation (quite explosive) Break down into smaller steps and store energy Chemiosmotic Theory Chemiosmotic Theory Links: 1. The use of high energy electrons to generate an electrochemical gradient Mitochondria : high energy electrons from oxidation of food Chloroplasts : harvesting light ….. 2. Utilize this electrochemical gradient to: Power molecular motors that drive ATP biosynthesis (ATPases) Drive transport of molecules against their concentration gradients. …. Common mechanism used across all organisms, eukaryotes, prokaryotes, etc … Chemiosmotic Theory Electrochemical Gradient in Mitochondria In mitochondria electrochemical gradient generated from protons. Electrochemical gradient () – units kJ mol-1 Chemical (pH) Difference in concentration of H+’s across the bilayer Electrical ( Separation of charge across the bilayer In bioenergetic is often defined as the proton motive force p with units of millivolts Electron Transport Chain  Overview of the Electron Transport Chain Each complex rich in redox centres zanov (2015) Nature Reviews in Molecular Cell Biology 16: 375- Electrochemistry : Revision Em : a measure of the tendency of a chemical species to acquire electrons and thereby be reduced. Large Em : high affinity for electrons Low Em : low affinity for electrons Em denotes the potential at which the compound is half oxidized and half reduced. DEm related to DG: ∆ 𝐺=− 𝑧𝐹 ∆ 𝐸 Therefore, electrons will move to sites with larger Em Nernst Equation If we know: ∆ 𝐺=− 𝑧𝐹 ∆ 𝐸 Then we can rewrite our equation for free energy in our system for a given mass action ratio: 0 ∆ 𝐺=∆ 𝐺 − 𝑅𝑇 ln ( [ 𝐴 ][ 𝐵 ] [ 𝐶 ][ 𝐷 ] ) In terms of the electrochemical potential: ∆ 𝐸=∆ 𝐸 +0𝑅𝑇 𝑧𝐹 ln ( [ 𝐴] [ 𝐵] [ 𝐶 ] [ 𝐷] ) So what redox centres do mitochondria utilize NAD+/NADH NAD+ 2 electron carrier + 2e- + H+ DEm~ -0.25 V NADH Redox Centres : Flavins 2 electron carrier +e- + H+ +e- + H+ DEm~ -0.20 V Redox Centres : Ubiquinone 2 electron carrier Membrane bound: large isoprenoid chain +e- + H+ +e- + H+ DEm~ 0.045 V  Redox Centres: FeS Centres 1 electron carrier (irrespective of number of Fe atoms) Linked via Cys or Cys/His Redox potential tuned by the protein DEm ~ -250 to 250 mV (in mitochondria) Redox Centres: Cytochromes 1 electron carrier Haem a Haem b Haem c DEm ~ 0.070 DEm ~ 385 mV mV DEm ~ 0.254mV The electron transport chain is an ordered series of redox centres The electron transport chain orders these redox centres according to their potential As electron make the small jumps for one redox centre to the next, the complexes of the ETC, couple this to the transport of protons. How does this coupling occur? Electron Transport Chain Complex I : NADH/UQ Oxidoreductase  Complex I: NADH/UQ oxidoreductase Thermus thermophilus Complex I zanov (2015) Nature Reviews in Molecular Cell Biology 16: 375-  Complex I: electron transfer zanov (2015) Nature Reviews in Molecular Cell Biology 16: 375- Transfer between redox centres Separation between redox centres large >10Å How do electrons travel such distances. Quantum Tunnelling DG∗ Energy Quantum Tunnelling Reaction Coordinate Separation between redox centres (Complex I).  Location of the UQ binding site Unusual facts: UQ binding site 15Å from the bilayer Hydrophilic in nature H+ from Tyr/His zanov (2015) Nature Reviews in Molecular Cell Biology 16: 375-  Complex I: Proton Pumping zanov (2015) Nature Reviews in Molecular Cell Biology 16: 375-  Proton Pumping/Electron Transfer Coupling Transverse helices: Stabilize complex Coordinate conf. change N2/UQ2- Provides energy for pumping Repulsion with acidic patch Four H+ translocated for every 2 electrons passing through the complex. One for each antiporter zanov (2015) Nature Reviews in Molecular Cell Biology 16: 375- Electron Transport Chain From NADH/Ubiquinone Oxidoreductase to Ubiquinone/Cytochrome c Oxidoreductase Complex III : Ubiquinone/Cyt c Oxidoreductase Cyt c1 cytochrome c1 Rieske Protein haem bH 2Fe2S – Rieske complex Cyt b haem bL The core of Complex III (1BE3.pdb) Q-Cycle : Mitchell’s Original Hypothesis 1st UQH2 2nd UQH2  Why do the electrons take different paths? Repositioning of Rieske protein, directs electrons to different redox centres. Electron Transport Chain From Ubiquinone/Cytochrome c Oxidoreductase to Cytochrome c Reductase Cytochrome c Charge + - Surface Charge Distribution Electron Transport Chain From Ubiquinone/Cytochrome c Oxidoreductase to Cytochrome c Reductase Complex IV : Cytochrome c Reductase 4 cyt c + 4 H+ + O2  2 H2O Transport of electrons to the a3/CuB centre  Reduction of O2 Occurs at binuclear centre (Cyt a3/CuB) Requires a conserved Tyr O2 binds to Cyt a3 Split between Cyt a3 and CuB Reduced by e- from Cyt c (P face), Whilst picking up H+ from N face Complex IV : Pumping protons K Channel: Protons for H2O D Channel: Proton pumping pathway  Summary: Electron Transport Chain zanov (2015) Nature Reviews in Molecular Cell Biology 16: 375- Summary Electron transport chain catalyses The transfer of electrons from NADH to O2 through a series of redox centres Couples the fall in electron energy to the movement of protons across the lipid bilayer. Results in the formation of an electrochemical gradient. Ensures that: Efficient coupling of electron transfer to H+ pumping. Number of ROS are minimised – reducing damage to cell. Question : If FAD was donating the electrons, from your knowledge of the ETC and the redox potentials, how would the electrons be introduced. Does such a complex exist? ATP Generation: F1F0-ATPase  F1F0-ATP Synthase F1F0-ATPase: F1 – site of ATP synthesis (a/b subunit) F0 – motor unit Couple Dp driven protein rotation with ATP synthesis Coupling between F1 and F0 by central stalk (g,d,e) Kühlbrandt (2019) Annual Reviews of Biochemistry 88:515-549 Structure of F1F0-ATPase Crystal structure of F1- ATPase Cryo-EM Structure of F1F0- ATPase Single particle analysis Improved detectors  Overview of the catalytic cycle Kühlbrandt (2019) Annual Reviews of Biochemistry 88:515-549 F0 Motor: utilizing Dp Composed of two types of subunit: a : stator, provide half channels for translocation of proton across the bilayer. c : rotor, 8 copies in mitochondria Role: Couple movement of protons across the bilayer to the rotation of the central stalk. The motor assembly From F1 c-subunits a-subunits Conserved Glu From side  The proton conduction pathway Kühlbrandt (2019) Annual Reviews of Biochemistry 88:515-549  The proton conduction pathway Kühlbrandt (2019) Annual Reviews of Biochemistry 88:515-549  Transmission to F1 domain via the central stalk Kühlbrandt (2019) Annual Reviews of Biochemistry 88:515-549 F1 Domain : the catalytic cycle 3a/b domains 1 ATP 1 ADP + Pi 1 Empty Conformational changes initiated by interaction with the g subunit g-subunit distorts nucleotide binding site Coupling rotation to ATP synthesis Three site alternating binding mechanism Rotation of g-subunit: i) Convert ATP site to open site, allowing release of ATP ii) Converts ADP/Pi into a tight binding site This allows: iii) The binding of ADP/Pi to the new empty site. iv) Conversion of ADP/Pi into ATP Events are cooperative, coordinated action between 6 subunits Summary F0 motor domain: Couples the movement of H+’s through the bilayer to the rotation of the c-ring Central Stalk: Propagates the rotational motion of the c-ring to the F1 catalytic domain F1 catalytic domain: Contains three a/b dimers arranged symmetrically around molecule. Central stalk breaks symmetry, perturbing the conformation of the ATP binding site, driving synthesis of ATP. References Molecular Biology of the Cell : Alberts et al., Chapter 14 – Energy Conversion: Mitochondria and Chloroplasts Bioenergetics 4 : Nicholls and Ferguson Lecture Specific Sazanov (2015) Nature Reviews in Molecular Cell Biology 16: 375- W. Kühlbrandt (2019) Annual Reviews of Biochemistry 88:515- 549 Walker (2013) Biochem Soc Trans 41:1-16 http://www.mrc-mbu.cam.ac.uk/projects/2248/molecular- animations-atp-synthase