Citric acid cycle Electron transport Citric acid cycle addendum to glycolysis it continues to oxidize pyruvate to carbondioxide The electrons obtained by oxidation of glycolytic substrates are ultimately transferred to oxygen.
central pathway that also serves to oxidize amino and fatty acids Fatty acids are broken down to acetyl-CoA and are used as a major energy source. can be considered the „hub“ of metabolic chemistry Overview of the citric acid cycle eight reactions of the citric acid cycle serve to convert acetyl- CoA into two molecules of CO2 energy released during that process is conserved in the:
three molecules of NADH one FADH2 one „high-energy“ compound (GTP) first recognized by Hans Krebs in 1937 Krebs cycle or tricarboxylic acid cycle In eukaryotes - in the mitochondrion All substrates and enzymes must be made in or transported to the mitochondrion intermediates are also intermediates of other pathways Example: oxaloacetate is used for gluconeogenesis The Citric Acid Cycle first intermediate of the cycle is citrate Synthesis of acetyl-coenzyme A The „fuel“ for the citric acid cycle is acetyl-CoA „high-energy“ thioester compound derived from the degradation of fatty acids and some amino acids end product of glycolysis – pyruvate- source for acetyl CoA Pyruvate is converted to acetyl-CoA by the pyruvate dehydrogenase multienzyme complex – 3 enzymes: • pyruvate dehydrogenase (E1) • dihydrolipoyl transacetylase (E2) • dihydrolipoyl dehydrogenase (E3) The pyruvate dehydrogenase multienzyme complex
In E.coli: The core of the complex is made of 24 E2 proteins forming a cube surrounded by 24 E1 and 12 E3 proteins advantage of multienzyme complexes:
• short travelling distance of substrates enhances reaction rates • substrate channeling minimizes loss of substrates due to side reactions • coordinated control of reactions Coenzymes and prosthetic groups involved in the pyruvate dehydrogenase reaction Enzymes of the citric acid cycle: 1. Citrate synthase Another example of acid-base catalysis one of the few enzymes that can form a carbon-carbon bond without the assistance of metal ion catalysis 2. Aconitase Aconitase catalyzes the reversible isomerization of citrate to isocitrate contains a so-called iron-sulfur complex - redox cofactor used in many biochemical transformations aconitase-catalyzed reaction is a redoxneutral isomerization iron-sulfur complex stabilizes a transiently occurring hydroxyl anion Loss of an iron inactivates the enzyme
3. Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to α- ketoglutarate produces the first CO2 and NADH in the citric acid cycle similar to the phosphogluconate dehydrogenase reaction in the pentose phosphate pathway. 4. α-Ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation of an α-keto acid producing the second CO2 and NADH of the citric acid cycle the second CO2 leaving the cycle is not derived from the acetyl-CoA that entered the cycle reaction is chemically identical to the pyruvate dehydrogenase reaction which produces acetyl-CoA. 5. Succinyl-CoA synthetase couples the cleavage of the „high-energy“ succinyl-CoA to the generation of a „high-energy“ nucleotide triphosphate Goes in 3 steps
6. Succinate dehydrogenase The remainder of the citric acid cycle is concerned with the reformation of oxaloacetate from succinate First of these „rebuilding“ reactions is the dehydrogenation of succinate to fumarate Succinate dehydrogenase is the only membrane-bound protein of the citric acid feeds the electrons of the reduced FAD directly into the electron transport chain 7. Fumarase Fumarase catalyzes the hydration of fumarate to malate 8. Malate dehydrogenase The last step of the citric acid cycle dehydrogenation of malate to recover oxaloacetate NAD+- dependent reaction reaction is very similar to the lactate and alcohol dehydrogenase reaction Endergonic Overview of ATP generation oxidation of one acetyl group releases 8 electrons which are used to reduce 3 NAD+ and 1 FAD molecule electrons are passed on to the electron transport chain where 3 molecules of ATP are generated per NADH and 2 per FADH2 total of 12 molecules of ATP are generated per turn of the citric acid cycle (24 per one molecule of glucose) Total - 38 molecules of ATP are produced under aerobic conditions from 1 molecule of glucose Regulation of the citric acid cycle Exergonic reaction steps are possible regulatory points citrate synthase, isocitrate dehydrogenase and α- ketoglutarate dehydrogenase are subject to regulation
Factors which regulate the activity of enzymes: 1. substrate availability 2. product inhibition 3. competitive feedback inhibition
Acetyl-CoA, citrate and succinyl-CoA act as product inhibitors ATP and succinyl-CoA act as competitive feedback regulators NADH plays a major role as a product inhibitor as well as a negative feedback regulator Relationships to other pathways Citric acid metabolites are also raw materials for biosynthetic reactions Example 1: oxaloacetate for gluconeogenesis Dual nature of cycle – described as amphibolic Example 2: acetyl-CoA which is required for fatty acid biosynthesis in the cytosol acetyl-CoA cannot cross the mitochondrial membrane - generated from citrate by the action of ATP-citrate lyase The glyoxylate cycle Plants - enzymes that allow the net conversion of acetyl-CoA to oxaloacetate can be used for gluconeogenesis „derivation“ of the citric acid cycle requires two additional enzymes: isocitrate lyase and malate synthase so-called glyoxylate cycle operates in two cellular compartments: the mitochondrion and the glyoxysome (specialized plant peroxisome) Net result - conversion of acetyl-CoA to glyoxylate instead of two CO2
The glyoxylate cycle is essential to germinating plant seeds glyoxysomes in germinating seeds - surrounded by lipid bodies contain triglycerides which are eventually degraded to acetyl- CoA converted to glyoxylate and further to oxaloacetate by means of malate synthase and malate dehydrogenase Oxaloacetate - used in the reactions leading to the net synthesis of glucose (gluconeogenesis) Summary Citric acid cycle Glyoxylate cycle
ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION metabolic oxidation of fuel compounds, such as glucose can be summarized as follows:
C6H12O6 + 6O2 -> 6CO2 + 6H2O
Released electrons are not directly transferred to molecular oxygen first transferred to produce NADH and FADH2 the process of metabolic fuel oxidation can be broken up in two „half“ reactions:
+ - C6H12O6 + 6 H2O -> 6 CO2 + 24 H + 24 e + - 6 O2 + 24 H + 24 e -> 12 H2O
NADH and FADH2 pass the electrons on to the electron- transport chain in the mitochondrial inner membrane Functions of the electron-transport chain NADH and FADH2 transfer electrons to an electron acceptor become reoxidized oxidized coenzymes reenter the substrate oxidation reactions of glycolysis and the citric acid cycle electrons are passed „down“ in a sequence of redox reactions (10 different redox centers in 4 protein complexes) - reduce molecular oxygen to water
During the electron transfer processes - protons are pumped across the inner membrane and generate a proton gradient
free energy of this electrochemical gradient - for the synthesis of ATP from ADP and phosphate
through oxidative phosphorylation The Mitochondrion Greek: mitos, thread + chondros, granule Contains: enzymes of the citric acid cycle (including pyruvate dehydrogenase), enzymes required for fatty acid oxidation enzymes and redox proteins involved in electron transport and oxidative phosphorylation referred to as the cell’s „power plant“ The Mitochondrion has about the size of a bacterium (0.5 x 1.0 μm) eukaryotic cell contains 2000 mitochondria - take up 20% of the cell’s volume has a smooth outer membrane “clefts” of inner membrane - site of the „respiratory activity“
Electron transport Most of the electron carriers are located in the inner mitochondrial membrane form complex integral membrane proteins electrons are passed from NADH with a redox potential of -0.315 V to redox centers of gradually more positive redox potential until the electrons end up on molecular oxygen Oxidation of NADH in mitochondria electrons from NADH - passed through 4 different protein complexes This breaks the free energy change into three smaller parcels Each contribute to the synthesis of ATP (oxidative phosphorylation) the oxidation of NADH yields approx. 3 ATP (under standard biochemical conditions) efficiency of the process is ~42%. under physiological conditions the efficiency is ~70%. Complex I: NADH-Coenzyme Q oxidoreductase
largest protein complex in the mitochondrial inner membrane consist of 43 polypeptides contains a flavin mononucleotide (FMN) Six-seven iron-sulfur clusters („non-heme iron“) Each iron is coordinated by four sulfur atoms – tetrahedral fashion The iron can undergo one electron reduction/oxidation (Fe3+/ Fe2+) FMN and CoQ can undergo one and two electron reduction/oxidation CoQ can move freely within the membrane (compared to FMN which is tightly bound) Complex II: Succinate-CoQ oxidoreductase
citric acid cycle enzyme succinate dehydrogenase transfers
the electrons to a covalently bound FAD to generated FADH2 In complex II, the electrons are then passed on to one [4Fe- 4S] cluster, two [2Fe-2S] clusters and one cytochrome b560 redox potential difference between succinate and CoQ is not sufficient to drive ATP synthesis However, complex II is important as it allows the entry of high-potential electrons into the electron-transport chain Cytochromes are electron-transport heme proteins are redox active proteins that contain a heme group heme-bound iron alternates between the ferric and ferrous state during electron transport In reduced states the various cytochromes have distinguishable absorbance spectra Complex III: CoQ-cytochrome c oxidoreductase
known as cytochrome bc1 passes the electrons from CoQ to cytochrome c contains two b-type cytochromes, one cytochrome c1 and one [2Fe-2S] cluster known as the Rieske center - bound to the iron-sulfur protein, ISP The Q-cycles Electrons from CoQH2 are transferred to cytochrome c in two so-called Q-cycles CoQ serves directly as the carrier of protons from the matrix to the intermembrane space two cycles pump two protons each (two reactions) Complex IV: Cytochrome c oxidase takes up the electrons from four reduced cytochrome c molecules Used in the reduction of one dioxygen molecule
contains four redox centers: heme a, heme a3, a copper atom (CuB) and a pair of copper atoms (CuA) Mechanism of cytochrome c oxidase Electron transfer in cytochrome c oxidase is linear Dioxygen binds near cytochrome a3 and is reduced to water
The four protons required to generate two molecules of water originate in the mitochondrial matrix Additionally, protons are translocated from matrix to the intermembrane space for every pair of electrons, two protons are pumped across the membrane
Oxidative phosphorylation free energy released by the electron-transport chain - stored in the electrochemical potential of the inner mitochondrial membrane potential is used by ATP-synthase (complex V) for the highly endergonic synthesis of ATP the electrochemical gradient is discharged by ATP-synthase and this exergonic reaction drives ATP synthesis (chemiosmotic theory) Theory- based on the believe that electron-transport results in the production of a „high energy intermediate“ whose breakdown yields ATP search for such an intermediate was unsuccessful The chemiosmotic theory Explains why:
Oxidative phosphorylation requires intact inner mitochondrial membranes The inner mitochondrial membrane is impermeable to H+, K+, OH- and Cl- (free diffusion of these ions would undo the electrochemical potential built up by the electron-transport chain) An electrochemical potential is measurable across the inner mitochondrial membrane Compounds that make the inner mitochondrial membrane - „uncouple“ electron transport from oxidative phosphorylation ATP synthase (complex V) membrane-bound multisubunit protein composed of two functional units F1 and F0 F0 is a water-insoluble transmembrane proton channel F1 on the other hand is a peripheral water-soluble protein composed of five types of subunits This multisubunit protein can be readily dissociated from the F0 part and is able of ATP hydrolysis but not of ATP synthesis The binding change mechanism generation of ATP by pumping of protons - broken into three phases
1. The F0 subunit translocates the protons 2. The F1 subunit carries out the synthesis of ATP from ADP and Pi 3. The physical interaction of the F0 and F1 subunit harnesses the exergonic transport of protons to the synthesis of ATP
In the so-called binding change mechanism the three αβ-subunits of the F1 unit exists in three different conformations:
• the L state binds the substrates loosely • the T state binds the substrates tightly • the O state binds the substrates very loosely or not at all Uncoupling of electron transport and oxidative phosphorylation Compounds that increase the membrane permeability of protons - give rise to uncoupling of electron transport and oxidative phosphorylation Uncouplers dissipate the proton gradient and disable ATP synthesis Lipophilic molecules that freely move in the membrane and hence shuttle protons across the membrane Summary Electron transport chain Complex I-IV Oxidative phosphorylation Binding-change mechanisms
QUIZ 3 Thursday 19.12.2019 10:00 BE ON TIME! Chapters: from midterm until today