CELL STRUCTURE AND FUNCTION TEST 2 REVIEW 1 GLYCOLYSIS AND THE CITRIC ACID CYCLE The four stages of glucose oxidation are 1 glycolysis the conversion in the cytosol of one 6 carbon glucose into two 3 carbon pyruvate molecules 2 citric acid cycle oxidation of pyruvate molecules into CO2 in the mitochondrion via a 2 carbon acetyl CoA intermediate 3 electron transport electrons transport chain generates a proton motive force by generateing a concentration gradient of H higher inside the intermembrane space 4 ATP synthesis ATP synthesis AKA oxidative phosphorylation in the mitochondrion The irreversible and rate limiting steps of glycolysis are Step 1 the conversion of glucose into glucose 6 phosphate Step 3 the conversion of fructose into fructose 1 6 bi phosphate Step 10 the conversion of phosphoenolpyruvate to 2 molecules of pyruvate After step one the process is no longer reversible Glycolysis takes place in the cytosol and is without oxygen Just for glycolysis there is a net gain of 2 ATP Substrate level phosphorylation is the formation of ATP from ADP and Pi catalyzed by cytosolic enzymes in reactions that do not depend on a proton motive force or molecular oxygen The role of substrate level phosphorylation is to generate ATP Even though substrate level phosphorylation does not have to have a proton motive force to occur it does require the addition in Steps 1 and 3 of 2 phosphates from 2 ATPs The reducing power of FAD and NAD PROTON MOTIVE FORCE The mitochondrial inner membrane 2 THE ELECTRON TRANSPORT CHAIN AND GENERATION OF THE There is a higher concentration of H in the intermembrane space Electrons flow through the inner membrane through four multiprotein complexes collectively called the electron transport chain In regards to the F0F1 complex the F0 lies in the inner membrane The F1 part lies in the matrix In the matrix there are ATP ADP antiporters There are also Pi OH antiporters as well With the proton transporters protons are transported against their concentration gradient in order to maintain pH and voltage across the inner membrane The electron transport complexes and function The electron transport chain causes the concentration gradient by pumping H out of the matrix and into the intermembrane space The energy to pump the H against their concentration gradient comes from the transport of e from NADH and FADH2 O2 yielding H2O The H concentration gradient and the electric gradient in the matrix and in the intermembrane space is stored energy called the proton motive force The electron transport chain is made up of 4 multiprotein complexes integral membrane subunits Complex I NADH CoQ reductase NADH is oxidized to NAD releasing 2e into the chain 4H are pumped out made of 40 subunits CoQ ubiquinone has 2H attached and shuttles 2e to complex III After donating them it becomes CoQ again and is recycled back to complex I Complex II Succinate CoQ reductase FADH from citric acid cylce is the source of 2e for this complex they are not as high energy as NADH Succinimate and CoQ become fumarate and CoQH2 No H transported and there are 4 subunits CoQ has 2H attached and shuttles 3e to complex III After donating 2e it becomes CoQ again and is recycled back to complex II Complex III CoQ Cytochrome c reductase After accepting 2e from CoQH2 it releases 2H into the intermembrane space 2e are picked up inside the complex by cytchrome c3 and then transferred to the oxidized form of it cytchrome c2 In addtion 2H not related to CoQH2 are pumped out into the intermembrane space also using the energy generated from the 2e Oxidized cytochrome c2 diffuses into internmembrane space and shuttles 2e to complex IV Complex IV Cytchrome c oxidase Cytchrome c2 reductase is reoxidized as it transports e to complex IV and then recycled back to complex III e are transferred one at a time to complex IV They are then finally passed to O2 by Cu yielding H2O Energy form this transfer is able to pump 2H again into the intermembrane space The malate shuttle Since the inner mitochondrial membrane is impermeable to NADH the malate shuttle is needed to shuttle electrons from the cytosolic NADH to NAD in the matrix These electrons are needed in the matrix to be transferred indirectly to O2 via the electron transport chain The malate shuttle works as follows Electrons from NADH in the intermembrane space are transferred to oxaloacetate forming a small molecule malate that can cross the inner mitochondrial membrane against its concentration gradient Then malate in the matrix is converted back to oxaloacetate reducing NAD to NADH Then oxaloacetate which cannot cross the inner mitochondrial membrane is converted to aspartate by the addition of an amino group from glutamate Then aspartate crosses the inner mitochondrial membrane to the intermembrane space going against its concentration gradient Then the aspartate is converted back to oxaloacetate and the process starts over again 3 HARNESSING THE PROTON MOTIVE FORCE FOR ENERGY REQUIRING PROCESSES The F0F1 ATP synthase The F0F1 ATP synthase is a multimeric protein complex bound to inner mitochondrial membranes thylakoid membranes of chloroplasts and the bacterial plasma membrane that catalyzes synthesis of ATP during oxidative phosphorylation and photosynthesis The ATP synthase is an F class pump It utilizes the proton motive force to synthesize ATP from ADP and Pi The protons move down an electrochemical gradient and move through the ATP synthase as they traverse the membrane What happens is when a proton moves in at the interface of the F0 part and the F1 part it drives the rotation of the ring made of c subunits This rotation is enough to cause a conformational change of the beta subunits The beta subunits are as follows the open state binds ADP and Pi weakly The loose state binds ADP and Pi more strongly The tight state binds ADP and Pi so strongly that they spontaneously form ATP When the ATP is formed energy is generated to changed the beta conformation which in turn rotates the gamma rod The rotation of the gamma rod has been proven by attaching an actin filament to it By doing this we are able to see it rotate 120 degrees every time the beta subunit changes Unlike glycolysis the production of ATP can be reversed The experimental evidence of the proton motive force and mediated ATP synthesis is in the following experiment Isolated chloroplast thylakoid vesicles containing F0F1 particles in a solution of 7 5 pH The pH was equal inside the vesicle and