• Transfer repeated card over here
• add- inhibotors of the passage of electrons to O2(Cyanide, carbon monoxide) blocks ATP synthesis
• and inhibition of ATP synthesis blocks electron transfer in mitochondria
• obligatory coupling

• When the flow of protons into the matrix through ATP synthase is blocked, no path exists for the return of protons to the matrix, and the continued expulsion of protons driven by the activity of the respiratory chain generates a large proton gradient.
• the proton motive force builds up until the free energy of pumping protons out of the matrix against this gradient equals or exceeds the energy released by the transfer of electrons  from NADH to O2
• at this point electron flow stops. the free energy for the overall process of electron flow coupled to proton pumping becomes 0 (delta G is 0/equlibrium).
• Therefore proton and electron transfer are inextricably linked!

• can disrupt membrane  with detergent or shear: electrons are still transferred to O2 but no ATP is made
• Chemically: DNP and FCCP are hydrophobic weak acids which diffuse across mitochondrial membranes
• after crossing the membrane these guys release a proton and mess with the proton gradient
• Chemically: Ionophores like Valinomycin allow inorganic ions to pass thru membranes, which uncouples electron transfer from oxidative phosphorylation by dissipating the electrical contribution to the electrochemical gradient

• because electron transfer in ATP synthesis is used to make the electrochemical potential, artificially creating a proton gradient replaces electron transfer in ATP synthesis
• Mitochondria manipulated to have a difference of proton concentration and a separation of charge, but no oxygen or any other oxidizable substrate still make ATP

• F1- peripheral membrane protein
• Fo (o for oligomycin sensitive) -integral to the membrane
• when F1 is removed from mitochondria membrane vesicles, membranes still have intact respiratory chains with Fo portion of ATP synthase. electrons can be transferred from NADH to O2 but no proton gradient can be formed. Fo lets in as many protons as are pumped out...no gradient
• Isolated F1 catalyzes ATP hydrolysis

ATP is Stabilized Relative to ADP on the Surface of F1---What drives the formation of ATP within the Active Site?

• ATP hydrolysis and condensation is reversible in F1ATPase
• Labeled 18O atoms in ATP catalyzed by F1ATPase show that water inserts oxygens at random in the released inorganic phosphate...when no energy is added to the system
• the delta G for this is 0 vs cytosolic reaction, which is -30.5
• ATP synthesis is favored because ATP is bound tightly enough to counterbalance the cost of making ATP. Binding affinit is much lower for ADP, which accounts for 40kj/mol binding energy difference, which drives the equilibrium towards ATP product formation
• what does this mean? less energy is needed when ATP is bound, because it is more stable in the active site environment than ADP. Because both species are equally likely to exist in the site, the fact that ATP is bound tightly means that it will be released less often...wait...arent we making ATP? ***Ask professor about this logic****

• In the absence of proton gradient no ATP is relesed from F1Fo complex
• Structure of molecule reveals that it must alternate between a form that bind ATP very tightl and one which releases ATP

• Three active sites of F1 take turns catalyzing ATP synthesis
• A given beta subunit starts in a conformation which binds ADP and Pi
• Subunit now changes conformation to one that binds and stabilizes ATP, bringing about the equilibration of ADP + Pi with ATP at the enzyme surface
• the last shift in conformation results in a conformation which has a low affinity for ATP
• Conformational changes are driven by proton passage through Fo Pore. shaft of epsilon and gamma subunits and cylinder of c subunit rotates, turning the three beta subunits
• one rotation of the gamma subunit causes each beta subunit to cycle through all of its conformatiosn and 3 ATP are released
• [image]
  

• NADH dehydrogenase of inner mitochondrial membrane can accept electrons only from NADH in the matrix
• shuttle systems carry reducing equivalents from cytosolic NADH into mitochondria by an indirect route...in liver/kidney/heart- malate/aspartate shuttle
• Reducing equivalents of cytosolic NADH are transferred to cytosolic oxaloacetate to yield malate (malate dehydrogenase)
• the malate formed passes through the inner membrane  via the malate-a-ketoglutarate transporter
• within the matrix the reducing equivalents are passed to NAD(+) by malate dehydrogenase. the resulting NADH can pass electrons to the respiratory chain---2.5 ATP's generated per pair of electrons thru chain
• oxaloacetate must then be recycled
[image]
• skeletal muscle and brain use another shuttle--glycerol 3-phosphate shuttle
• this one delivers reducing equivalents from NADH to ubiquinone-complex III- not I, providing enough energy to generate 1.5 ATP per pair of electrons

• Proton motive force also drives several other processes essential for oxidative phosphorylation
• inner mitochondrial membrane has transport systems which shuttle ADP and Pi into the matrix and ATP out to the cytosol
• adenine nucleotide translocase-inner membrane- binds ADP(3-) in exhange for an ATP(4-), which is xported outward
• net negative charge moved out makes this electrochemically favorable and gives the matrix a net negative charge
• the proton motive force essentially allows for ADP-ATP exchange
• phosphate translocase-symport of H2PO4 and one proton into the matrix- also favored by the proton gradient- one proton is taken from high to low concentration side... using some of the energy of the electron transfer
•  [image]