Major concepts

1. What process provides the energy for ATP synthesis by ATP synthase? How is that process related to electron transport?
2. How does F o move protons across the membrane?
3. How are the conformation changes of F₁ (in ATP synthase) related to the function of F_o?
4. What determines the stoichiometry of ATP synthesis? What is the relationship between NADH oxidation and ATP synthesis, and why doesn't it have to be a whole-number relationship? What is the relationship between QH₂ oxidation and ATP synthesis?
5. Why are shuttles required for recycling NADH produced in glycolysis? How are they related to your answers to question 4?
6. What type of regulation is used for oxidative phosphorylation?

Core knowledge

1. What are the important subunits of F_o in ATP synthase? What does each do in order to move H⁺ across the membrane?
2. What are the important subunits of F₁ in ATP synthase? What does each do?
3. What are the conformations of the β subunits, and what are their characteristics?
4. What carrier moves protons across the membrane P → N, and what other molecule is moved at the same time? What other carriers are required for ATP synthesis? What carriers and enzymes are required for NADH shuttles? Which carriers are antiports, and which are symports?
5. What is the net number of ATP synthesized per glucose degraded to CO₂? How does that number change when cell type changes?
6. What are the important molecules that are involved in regulation of oxidative phosphorylation?

ATP synthase = F-type ATPase
A. structure =
     F_o stalk: integral membrane protein with 3 types of subunits = a, b, c (ab₂c_10-14δ)
         Each c = 2 transmembrane helices; the c's form a double circle through the membrane.
               The circle rotates clockwise within the membrane.
              Each c has an Asp side chain (that can accept H⁺) exposed to the membrane.
          The a subunit = 2 half channels, one exposed to the P side and one to the N side
               There is no passage from one half channel to the other; both are open to Asp on c.
               Moving clockwise, the N half channel is before the P half channel.
     F₁ portion: peripheral membrane protein in the matrix with α₃β₃γδε
         α and β subunits alternate, while γδε connect the β subunits to F_o.
         β subunits have the active sites, in three conformations= loose, tight, and open
              loose: binds ADP + P_i (moderate ADP affinity)
              tight: binds ADP + P_i tightly → ATP (high affinity for ATP)
              open: releases ATP (low affinity for ATP
B. function of F_o:
      1. When the c Asp with H⁺ (= –COOH) is exposed to the N side half channel, H⁺ leaves (→ matrix).
          The Asp is now Asp⁻ (–COO⁻), and exposing the (−) to nonpolar lipids is very
               unfavorable, so the only favorable rotation is clockwise.
      2. Rotation exposes the c Asp⁻ to the P side half channel.
          H⁺ entering the half channel from the P side can protonate the Asp⁻ → Asp⁰.
      3. The c subunit can now rotate into the bilayer, and a different Asp contacts the N side
          half channel. The H⁺ on the second Asp exits to the N side, and the process repeats.
      4. Movement of the c ring is coupled to rotation of γ. γ is linked to F₁ (does not rotate).
C. function of F₁:
     1. each β subunit starts in a different conformation (one loose, one tight, one open);
          no change is possible until loose has ADP + P_i bound
      2. movement of H ⁺ through the channel causes rotation of c subunits → γ rotation
         causing change in conformation of β subunits:
     3. tight → open, loose → tight, and open → loose
         ATP is released from open, ADP + P_i → ATP in tight, and open → higher affinity
     4. best accepted ratio is 3 H⁺ moved through ATP synthase/ATP synthesized
         Each complete rotation of c ring → 3 ATP synthesized;
         With 10 c subunits, that is 10 H⁺ moved/rotation, or ∼ 3 H⁺ moved for each ATP.

Movement of molecules across the inner membrane requires transporter proteins.
1. adenine nucleotide translocase
      ADP³⁻/ATP⁴⁻ antiport moves ADP and ATP down their concentration gradients
      favored direction = ATP out (more negative charges moving toward + side) and ADP in
2. phosphate translocase
     H₂PO₄⁻ /H⁺ translocase = symport that moves P_i into matrix (unfavorable)
         using H⁺ concentration gradient (favorable)
3. NADH shuttle uses two transporters
     a. malate/α-ketoglutarate transporter = antiport, moving malate from cytosol → matrix
     b. aspartate/glutamate transporter = antiport, moving aspartate from matrix → cytosol
     c. cytosol reactions: Asp + α-ketoglutarate ↔ oxaloacetate + glutamate
              oxaloacetate + NADH + H⁺ ↔ malate + NAD⁺
     d. matrix reactions: malate + NAD⁺ ↔ oxaloacetate + NADH + H⁺
              oxaloacetate + glutamate ↔ aspartate + α-ketoglutarate
     e. effect: moves NADH produced in glycolysis into the matrix for electron transport
4. glycerol-3-phosphate shuttle uses cytosol NADH to convert DAP → glycerol-3-P
     mitochondrial membrane glycerol-3-phosphate dehydrogenase catalyzes
      glycerol-3-P → DAP and reduces FAD → FADH₂
     FADH₂ is recycled by reducing Q, like succinate dehydrogenase.
     Found in skeletal muscle and brain.

Overall H⁺/ATP ratio = 4 H⁺ moved/ATP synthesized, so each NADH = 2.5 ATP
and each succinate (FADH₂) = 1.5 ATP
For malate/aspartate shuttles:
      1 glucose = 2 ATP + 2 NADH (glycolysis) + 2 NADH (pyruvate → AcCoA) +
         6 NADH + 2 FADH₂ + 2 GTP (citric acid cycle) = 32 ATP
For glycerol-3-phosphate shuttle:
     NADH produced in glycolysis = FADH₂ in the mitochondrial membrane, so
     1 glucose = 2 ATP + 2 FADH₂ + 2 NADH + 6 NADH + 2 FADH₂ + 2 GTP = 30 ATP

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