Biochemistry And Cell Biology - Bioenergetics PDF

BS31004 Biochemistry and Cell Biology Bioenergetics Marios Stavridis Intended learning outcomes An understanding of the structure and function of mitochondria An appreciation of the central principles of chemiosmotic theory Detailed knowledge of the structure, function, and mol...

BS31004 Biochemistry and Cell Biology Bioenergetics Marios Stavridis Intended learning outcomes An understanding of the structure and function of mitochondria An appreciation of the central principles of chemiosmotic theory Detailed knowledge of the structure, function, and molecular mechanism of the respiratory electron transport chain Knowledge of the molecular basis of ATP synthesis for the cell and a clear understanding of how this is coupled to the activity of the respiratory electron transport chain  References Alberts et al., Molecular Biology of the Cell (6th edition); Garland Science (available in library but do not hoard it) Chapter 14, pp753-782. Older editions are still OK (my old 3rd edition from 1994 perhaps even better for this chapter!). 4th edition free from PubMed Books –see reading lists link on My Dundee Nicholls and Ferguson, Bioenergetics (4th edition); Academic Press (available online within UoD network: https://www.sciencedirect.com/book/9780123884251/bioenergetics ) various references within this ppt –I think a bit too much on the detailed side… Alberts et al., Essential Cell Biology (3rd edition); Garland Science (available in library) Chapter 14 (a simpler version of MBotC) Stryer Biochemisty (7th edition); Freeman (available in library) Chapter 18 (in my 1995 4th edition it is chapter 21) 3 The Structure of the Mitochondrion 1. ATP synthesis 2. TCA Cycle 3. β-oxidation 4. Urea Cycle 4 The Structure of the Mitochondrion Outer membrane Inner membrane Intermembrane space Matrix outer membrane 5 nm IMS 10 nm inner membrane 5 nm 5  The Structure of the Mitochondrion 400-500 different proteins located in a mitochondrion Mitochondrial ribosomes (55S) Mitochondrial genome  (16.5 kbp, 22 tRNAs, 2 rRNAs, 13 ORFs) 6  Biogenesis of the Mitochondrion Mnf1 and 2 = ‘mitofusin’ proteins OPA1 = optic atrophy 1 protein Drp1 = cytoplasmic GTPase triggered by ER fusion. It forms a ring that then constricts. Mutations in mfn2 and OPA1 can lead to a form of Charcot-Marie-Tooth neuropathy. 7 Degradation of the Mitochondrion - mitophagy 8 Energy Transduction IMS positive acid H+ H+ H+ H+ H+ H H+ H+H+ H+ H+ H+ + inner H+ + Δ ΔpH H OH-OH- OH- OH- OH- OH- OH- OH- OH- negative alkali matrix PROTON ELECTRO -CHEMICAL GRADIENT Δ = Vmatrix- VIMS Δ ~ -200 mV ΔpH = pHmatrix – pHIMS ΔpH ~ 0.5 9 How much free energy is in a proton Δ ~ -200 mV gradient? ΔpH ~ 0.5 IMS acid H+ H+ H+ H+ H+ H H+ H+H+ H+ H+ H+ positive + inner H+ + Δ ΔpH H OH-OH- OH- OH- OH- OH- OH- OH- OH- negative alkali matrix PROTON ELECTRO -CHEMICAL GRADIENT FOR ANY CHEMICAL GRADIENT: G = R  T  ln ([inside]/[outside]) Gas constant ‘R’ = 8.3 kJ mol-1 T = absolute temperature (K) 10  NOTE I have hidden the next set of slides because I do not want to spend time in class to explain them. This is very basic re-arranging of equations, substitutions etc. If you need help working it out, ask Prof. Google, Dr. Bing or Ass. Prof. Yahoo. You should be able to use these equations to calculate differences in electrochemical gradient energy with different concentrations and charges of ions. If you understand the properties of logarithms and what pH means then you should be fine. Don’t worry about memorising this or if you get slightly different ways to express it on the web. I have tried to keep all the “Delta” differences consistent (difference of inside minus outside) so the signs may appear different from other places (and older versions of this ppt) If you want to work it out, MIND THE UNITS! MPS 11 How much free energy is in a proton gradient? Δ ~ -200 mV ΔpH ~ 0.5 IMS acid H+ H+ H+ H+ H+ H H+ H+H+ H+ H+ H+ positive + inner H+ + Δ ΔpH H OH-OH- OH- OH- OH- OH- OH- OH- OH- negative alkali matrix PROTON ELECTRO -CHEMICAL GRADIENT FOR ANY ELECTRICAL GRADIENT: G = n  F  Δ n = charge of ion to move Faraday constant F = 96.5 kJ mol-1 V-1 14 How much free energy is in a proton gradient? Peter Mitchell simplified this equation to explain his theory of “proton motiveforce” occurring at 37 C in a mitochondrion: p (mV) = -Δ + 61pH (some books say p (mV) = -Δ + 59pH !) 17 Respiration H+ H+ outside acid positive ionically sealed membrane ΔpH Δ inside reductase / alkali negative dehydrogenase ‘oxidase’ S-H S + H+ + 2e- S + H+ + 2e- S-H (electron donor) (electron acceptor) Put very simply, oxidation of reduced substrates, linked to reduction of oxidised substrates, generates a proton and voltage gradient …. it’s obviously quite a bit more complicated than this …. proton motive force = Δp (mV) = Δ – 59(or 61)ΔpH 18  (most likely to Strongest Redox couples E0' (Volts) release electrons) reductants 2H+/H2 (- 0.42 V) Redox potentials - 0.4 and electron NAD+/NADH (- 0.32 V) transfer from - 0.2 electron donors to SO42-/H2S (- 0.22 V) electron acceptors Fumarate/succinate + (0.03 V) 0.0 Cyt box/Cyt bred + (0.04 V) UQox/UQred + (0.10 V) NADH + H+ + ½O2 + 0.2 Cyt cox/Cyt cred + (0.25 V) ® NAD+ + H2O NO2-/NH3 (+ 0.34 V) (E0' = 1.14 V) Cyt aox/Cyt ared + (0.39 V) NO3-/NO2- (+ 0.42 V) + 0.4 common in + 0.6 aerobic respiratory Fe3+/Fe2+ (+ 0.77 V) e- transfer ½O2/H2O (+ 0.82 V) + 0.8 (most likely to take Strongest up electrons) oxidants 19 Respiration what compounds can be used /+60* *Nicholls & Ferguson (2013) Bioenergetics4 (AP 2013) 20  Respiration How are electrons transferred? within proteins – cofactors FLAVINS 21 flavins can carry 2 electrons at a time Respiration How are electrons transferred? within proteins – cofactors rubredoxin 2Fe-2S 4Fe-4S ‘cubane’ 22 Fe-S clusters can carry 1 electron at a time  Respiration haem / heme How are electrons transferred? within proteins – cofactors 23 haems can carry 1 electron at a time Respiration How are electrons transferred? outwith and between proteins - in the lipid bilayer  Ubiquinone  Menaquinone (Vit K2)  Demethylmenaquinone menaquinone demethylmenaquinone 24 quinones can carry 2 electrons at a time Respiration Cofactors have their own redox potentials 25 Respiration – the mitochondrion Glycerol-3-P GlpD Fatty acids ETF a mitochondrion quinone pool ‘Complex I’ ‘Complex III’ NADH Succinate ‘Complex II’ ‘Complex IV’ Oxygen compound enzyme enzyme compound 26  NOTE I have hidden the next set of slides because I do not want to spend time in class to explain them. The key concept I need you to understand is in the next visible slides but if you want to understand this better then go through the hidden slides in your own time and try to see how each step works MPS 27  Respiration – the mitochondrion Glycerol-3-P GlpD ATP synthesis is the major route of completing the proton circuit 3 /13 mV 5/60 +4 +220 mV -320 +820 mV +30 mV mV Respiration How many protons to make an ATP? Work out the P:O ratio  How many ATPs are made per O2 burned?  Classical experiments done in a Clark O 2 electrode 1 O2 ~5-6 ATP produced 1 O2 20 H+ pumped ~3-4 H+ per ATP Respiration How many protons to make an ATP? thermodynamics  How much free energy in ATP hydrolysis? -50 kJ mol-1 thus, 50 kJ mol-1 need to synthesise ATP each proton releases -22 kJ mol-1 ~2.2 H+ per ATP Why the extra proton? The mitochondrion makes ATP for the cell, not for itself ADP 2 negative charges in IMS positive acid H+ H+ H+ H+ H+ H+ H+ H+ H+ inner H+ + ANT Δ ΔpH H alkali OH-OH- OH- OH- OH- OH- OH- OH- negative matrix ATP 3 negative charges out The Adenine Nucleotide Exchanger is driven by Δ, but pH is unaffected Why the extra proton? The mitochondrion makes ATP for the cell, not for itself IMS positive acid H+ H+ H+ H+ H+ H+ H+ H+ H+ inner H+ + Δ ΔpH H alkali OH-OH- OH- OH- OH- OH- OH- OH- negative matrix OH- A phosphate (H2PO4-) / hydroxide antiporter The phosphate carrier is driven by pH, but Δ is unaffected Why the extra proton? The mitochondrion makes ATP for the cell, not for itself IMS positive acid H+ H+ H+ H+ H+ H+ H+ H+ H+ inner H+ ANT Δ ΔpH H+ OH-OH- OH- OH- OH- OH- OH- OH- negative alkali matrix OH- One extra proton must be translocated to support ATP supply to the cell “Uncoupling” Dinitro phenol (DNP) Carbonyl cyanide m-chlorophenyl hydrazone “Uncoupling” – brown fat mitochondria UNC1 H+ Uncoupling Protein 1 (UNC1) 32 kDa integral membrane protein “Uncoupling” – brown fat mitochondria Uncoupling Protein 1 (UNC1) 32 kDa integral membrane protein Fedorenko et al. (2012) Cell 151:400-413.  Summary Energy production follows thermodynamic rules and involves complex biochemistry Electron flow powers proton pumps that generate chemiosmotic gradient Quinones and other intermediates facilitate electron flow Electrons need to be transferred to terminal acceptor Chemiosmotic gradient is a dynamic equilibrium: production (H+ pumps) and dissipation (ATP synthase) These principles also apply to Photosynthesis and some aneaerobic metabolism (see other lectures in this module) The precise details vary depending on the system you study 49