Krebs Cycle and Oxidative Phosphorylation YR1 Lecture 1H 2023 PDF

Krebs cycle and Oxidative Phosphorylation Dr Bronwen Dalziel COPYRIGHT COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been reproduced and communicated to you by or on behalf of University of Western Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The m...

Krebs cycle and Oxidative Phosphorylation Dr Bronwen Dalziel COPYRIGHT COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been reproduced and communicated to you by or on behalf of University of Western Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice. Lecture Outline The Krebs cycle (TCA cycle) continues the journey of converting glucose to energy after glycolysis. NADH and FADH2 from the Krebs cycle are used to transport energy to the electron transport chain (oxidative phosphorylation) ATP is made by ATPase when protons (H+) cross the inner membrane of the mitochondria. Glucose C-C-C-C-C-C glycolysis Glycolysis C-C-C C-C-C Pyruvate 2 ATP 2 NADH Image: By NEUROtiker (Own work) via Wikimedia Commons Krebs cycle (Citric Acid Cycle) glycolysis Understanding Pathophysiology, ANZ Edition, Chapter 3, 32-57 Preparatory step for Krebs cycle Outer compartment C O2 Coenzyme A C C C Pyruvate NAD+ Matrix CoA C C Acetyl CoA NADH Pyruvate dehydrogenase complex Now ready for the Krebs cycle! Energy score so far: 1 NADH Dietary deficiency of thiamine (Vit B1) allows pyruvate to accumulate as it is a coenzyme for pyruvate dehydrogenase which is the first step in getting the pyruvate ready for the Krebs cycle. Many alcoholics are thiamine deficient (both because of a poor diet and also because alcohol inhibits thiamine absorption) and may develop potentially fatal pyruvic and lactic acidosis. Patients with inherited pyruvate dehydrogenase deficiency, which can be the result of defects in one or more of the components of the enzyme complex, also present with lactic acidosis, particularly after a glucose load. Because of the dependence of the brain on glucose as a fuel, these metabolic defects commonly cause neurological disturbances. Krebs cycle Acetyl CoA C-C Citric acid C-C-C-C-C-C Oxaloacetic acid C-C-C-C CO2 1 NADH NADH NADH FADH2 CO2 NADH Energy score so far: 1 ATP 3 NADH 1 FADH2 ATP CO2 Summary: The acetyl molecule enters the krebs cycle by combining with oxaloactetic acid to form citric acid (hence the name citric acid cycle). The molecule is progressively oxidised through a series of enzyme catalysed reactions to form CO2 and 1 ATP molecule, while NAD+ and FADH are reduced to form 3 NADH, 1 FADH2. Remember this cycle happens twice for each glucose molecule (there are 2 pyruvates at the end of glycolysis). See next slide Krebs cycle Acetyl CoA C-C Citric acid C-C-C-C-C-C Oxaloacetic acid C-C-C-C CO2 2 NADH NADH FADH2 NADH 2x CO2 NADH Energy score so far: 2 ATP 6 NADH + 2NADH (prep) 2 FADH2 ATP CO2 Krebs cycle Acetyl CoA C-C Citric acid C-C-C-C-C-C Oxaloacetic acid C-C-C-C NADH Fatty Acids NADH FADH2 CO2 NADH ATP CO2 Nice to know now but the details are in Dr Victoria Gauci’s lecture on Fat Metabolism. The breakdown of fatty acids, called fatty acid oxidation or beta (β)-oxidation, begins in the cytoplasm. Fatty acids are transported into the mitochondrial matrix as fatty acyl carnitine. Once inside the mitochondrial matrix, the fatty acyl carnitine molecule is converted back into fatty acyl CoA and then into acetyl CoA. The newly formed acetyl CoA enters the Krebs cycle and is used to produce ATP in the same way as acetyl CoA derived from pyruvate. Pyruvate C-C-C Amino Acids Acetyl CoA C-C Krebs cycle Oxaloacetic acid Citric acid C-C-C-C-C-C C-C-C-C NADH NADH FADH2 Amino Acids CO2 NADH Fumarate ATP CO2 ↑ATP CO2 Acetyl CoA C-C Oxaloacetic acid C-C-C-C 1 Krebs cycle Citric acid C-C-C-C-C-C NADH NADH ↑ADP FADH2 2 CO2 NADH 1 = citrate synthetase 2 = isocitrate dehydrogenase ATP CO2 Regulation ↑[ATP] inhibits citrate synthetase from making citric acid (accepting acetyl group from CoA) ↑[ADP] activates isocitrate dehydrogenase to speed up the cycle Toxins in the Krebs cycle Fluoroacetyl-CoA Fluoroacetate Rodent kill – ‘compound 1080’ Metabolised to fluoro-citrate (enzyme inhibitor) which blocks Krebs cycle completely Fluorocitrate Oxaloacetic acid Toxins in the Krebs cycle Fluoroacetyl-CoA Fluoroacetate Arsenic can disrupt glycolysis, pyruvate and succinate oxidation pathways by binding to inorganic phosphate depleting ATP stores Fluorocitrate Oxaloacetic acid ATP Story so far We have 2 ATP from glycolysis 2 ATP from the Krebs cycle need more ATP please…. ….Oxidative Phosphorylation We have 10 NADHs and 2 FADH2s Breathe Oxidative Phosphorylation Inner mitochondrial membrane Outermembrane Outer compartment III I IV Inner Membrane Matrix NAD+ NAD - H H+ H+ H+ Outermembrane H+ H+ H+ H+ H H+ H+ + H+ H+ H+ I III - Outer compartment IV Inner Membrane Matrix NAD+ 1/ O 2 2 + 2H+ = H20 Outermembrane H H+ + H + H+ H+ H+ H+ H+ H+ H+ Outer compartment I III IV Inner Membrane ATP synthase Matrix ADP àATP 1 NADH = 3 ATP Outermembrane H H+ + H I FADH2 II - - III + H+ H+ H+ IV Outer compartment Inner Membrane Matrix FAD 1 FADH2 = 2 ATP Outermembrane H H+ H H+ I II H+ + III + H+ H+ H+ IV H+ Outer compartment Inner Membrane Matrix 2 x FADH2 10x NADH 1 NADH = 3 ATP x10 = 30 ATP 1 FADH2 = 2 ATP x2 = 4 ATP Total ATP from Glucose = 30 + 4 + 2 glycolysis + 2 Krebs = 38 ATP -> 30ATP due to transport of molecules across membranes and loss of protons across membranes Regulation ↑[ATP] inhibits citrate synthetase from making citric acid (accepting acetyl group from CoA) ↑[ADP] activates isocitrate dehydrogenase to speed up the cycle The most important factor in determining the rate of oxidative phosphorylation is the level of ADP. The rate of oxygen consumption by mitochondria increases markedly when ADP is added and then returns to its initial value when the added ADP has been converted into ATP (i.e. the ETC is coupled to the utilisation of ATP and will speed up as ATP is used up) The availability of NADH, FADH2 and O2 and ADP drives the ETC/oxidative phosphorylation Regulation ↑[ATP] inhibits citrate synthetase from making citric acid (accepting acetyl group from CoA) ↑[ADP] activates isocitrate dehydrogenase to speed up the cycle The most important factor in determining the rate of oxidative phosphorylation is the level of ADP. The rate of oxygen consumption by mitochondria increases markedly when ADP is added and then returns to its initial value when the added ADP has been converted into ATP (i.e. the ETC is coupled to the utilisation of ATP and will speed up as ATP is used up) The availability of NADH, FADH2 and O2 and ADP drives the ETC/oxidative phosphorylation Regulation Oxygen is vital in oxidative phosphorylation to accept electrons and form water. If there is no oxygen, then complex IV remains reduced and, without getting NAD+ back, Krebs can’t continue Outermembrane H H H H H Outer compartment -I - -IV III - Inner Membrane Matrix NAD - H - 1/ O 2 2 + H+ = H20 ADP àATP Krebs cycle CO2 Acetyl CoA C-C Citric acid C-C-C-C-C-C Oxaloacetic acid C-C-C-C NADH NADH FADH2 CO2 NADH ATP CO2 Clinical insight Carbon monoxide poisoning (e.g. natural gas or car exhaust fumes in a closed space) – CO displaces O2 in your RBCs – With no O2 the ETC and Krebs come to a halt – Glycolysis alone is not enough for our bodies Eventually leads to – Permanent brain damage – Damage to your heart, possibly leading to life-threatening cardiac complications – Foetal death or miscarriage – Death Regulation ↑[ATP] inhibits citrate synthetase from making citric acid (accepting acetyl group from CoA) ↑[ADP] activates isocitrate dehydrogenase to speed up the cycle The most important factor in determining the rate of oxidative phosphorylation is the level of ADP. The rate of oxygen consumption by mitochondria increases markedly when ADP is added and then returns to its initial value when the added ADP has been converted into ATP (i.e. the ETC is coupled to the utilisation of ATP and will speed up as ATP is used up) The availability of NADH, FADH2 and O2 and ADP drives the ETC/oxidative phosphorylation Krebs cycle creates some heat…. So uncoupling oxidative phosphorylation = heat (thermogenesis) which is useful in babies. Skeletal muscles are the biggest source of heat in our bodies because they consume the most ATP proportionally to other organs Outermembrane H+ H+ H+ H+ H+ Outer compartment I II IV Inner Membrane UCP NAD - H - 1/ O 2 2 + H+ = H20 ADP àATP Matrix Summary Krebs cycle is used to reduce molecules of NAD+ and FAD as well as make some ATP Oxidative phosphorylation uses the NADH and the FADH2 to drive protons across the inner mitochondrial membrane so that they flow back through ATP synthase to make ATP One glucose molecule can make 38 ATPs – 2 from glycolysis, 2 from the Krebs cycle, 30 from NADHs and 4 from FADH2s (BUT = 36 NET as 2 ATPs are used in transport of NADH from glycolysis into mitochondria) ATP is used as a way to store instant energy in our cells Questions: Dr Bronwen Dalziel Senior Lecturer Medical Education [email protected]