Outline

7.1 The citric acid cycle
7.2 The electron transport chain
7.3 ATP biosynthesis

Learning Objectives

1. Describe how acetyl-CoA is oxidized and other molecules are transformed in the citric acid cycle.
2. Illustrate how electron transport results in protons ($H^{+}$) getting pumped out of the mitochondrial matrix, and the formation of water.
3. Describe how the cell uses the electrochemical gradient produced in electron transport to synthesize ATP.

Section 7.1 Learning Objective

Describe how acetyl-CoA is oxidized and other molecules are transformed in the citric acid cycle.

The Citric Acid Cycle

Also known as the Krebs cycle and tricarboxylic acid cycle
Eight reactions that serve as a central metabolic hub
Generates the following:

• 2 $C{O}_2$
• 1 GTP or ATP
• 3 $NADH$
• 1 $FAD{H}_2$
  Example of a cyclic pathway
  Electrons as $NADH$ and $FAD{H}_2$ for use in electron transport
  1 $NADH$= 2.5 ATP and 1 $FAD$ = 1.5 ATP

The Citric Acid Cycle

Figure 7.2 Citric acid cycle.

Example of a metabolon

Metabolon Defined

Metabolon is a group of enzymes performing reactions with a common purpose.
Enzymes can be localized in an organelle or part of an organelle.
Associated with glycolysis, glycogenolysis, fatty acid biosynthesis, and the electron transport
Substrate channeling may occur.

Substrate Channeling Defined

Substrate channeling is the diversion of the product of one enzymatic reaction directly into a subsequent reaction, to increase reaction rate and efficiency.

Acetyl-CoA

Important intermediate in several pathways
Product of fatty acid catabolism
Chemical bonds are energetically rich.

Citrate Synthase

Condensation of acetyl CoA with oxaloacetate
Produces citrate

Aconitase

Isomerization of citrate to isocitrate
Reversible reaction
Produces citrate

Isocitrate Dehydrogenase

Oxidation of isocitrate to α-ketoglutarate
^+$+ is reduced.
Produces citrate

Please point of the for the TCA cycle: $NADH$is a product for 3 of the 4 dehydrogenases and inhibits 3 of these enzymes

α-Ketoglutarate Dehydrogenase

α-ketoglutarate is oxidized to succinyl-CoA.
Rate-determining step
^+$+ is reduced.

α-Ketoglutarate Dehydrogenase Complex

Multienzyme complex
Three different subunits:

• E1—TPP decarboxylase
• E2—dihydrolipoyl transferase
• E3—dihidyrolipoyl dehydrogenase

Succinyl CoA Synthetase

Substrate-level phosphorylation

• Nucleotide is generated directly by phosphorylating a nucleoside
  Reversible reaction
  Produces succinate and ATP or GTP
  Two isoforms of enzyme – one form will produce GTP and the other will form ATP.
  

Succinate Dehydrogenase

Oxidation of succinate
FAD is reduced.
Produces fumarate, a trans-dioic acid

FAD is tightly bound to the enzyme and quickly passes these electrons to ubiquinone to reduced ubiquinone in the electron transport chain.

Fumarase

Hydration reaction
Reversible reaction
ATP is neither consumed nor produced.

Malate Dehydrogenase

Oxidation reaction
Reversible reaction
$NA{D}^{+}$ is reduced.

A Schematic View of the Citric Acid Cycle

Figure 7.3 Schematic view of the citric acid cycle.

Energetics of the Citric Acid Cycle

Regulation of the Citric Acid Cycle

Based on substrate availability

• Increased levels of substrates generally increase flux through the pathway
  $NADH$ inhibits three dehydrogenases.
  Allosteric regulators:
• ATP
• $C{a}^{2+}$
• Succinyl CoA

Regulation of the Citric Acid Cycle

Figure 7.4 Regulation of the citric acid cycle.

Anaplerotic Reactions

Anaplerotic reactions replenish levels of citric acid cycle intermediates through a number of mechanisms.

Figure 7.5 Central role of the citric acid cycle in metabolism.

Transamination Reactions

Transamination reactions occur when amine groups are shuttled to α-ketoglutarate to generate glutamate and a new α-keto acid from the old amino acid.

α-Ketoglutarate Replenishment

Levels of α-ketoglutarate can be replenished by removal of the amine moiety from the amino acid glutamate.

Succinyl-CoA Replenishment

Propionyl-CoA can be generated via catabolism of odd-chain fatty acids, branched-chain amino acids (Leu, Ile, Val), or the amino acids Thr and Met (via α-ketobutyrate)

Figure 7.6 Conversion of propionyl-CoA to succinyl-CoA.

Propionyl CoA Carboxylase Mechanism

Figure 7.7 Mechanism of propionyl-CoA carboxylase.

Methyl Malonyl CoA Mechanism

Figure 7.8 Mechansim of methyl malonyl-CoA mutase.

Oxaloacetate Replenishment

Can be produced from pyruvate through gluconeogenesis
Retained in the mitochondrial matrix for use in the TCA cycle
Can be generated from glutamate via aspartate transamination
Pyridoxal phosphate is a cofactor

Figure 7.10 Transamination of aspartate.

Pyruvate Carboxylase Mechanism

Figure 7.9 Mechanism of pyruvate carboxylase.

Section 7.2 Learning Objective

Illustrate how electron transport results in protons ($H^{+}$) getting pumped out of the mitochondrial matrix, and the formation of water.

Reducing Equivalents

Figure 7.11 Reducing equivalents come from multiple metabolic pathways.

Through these processes – 4 ATP molecules and 10 $NADH$ and 2 $FAD{H}_2$ molecules are generated from a single molecule of glucose.

Electron Transport Chain

Occurs in the matrix and inner mitochondrial membrane

Figure 7.12 Overview of the electron transport chain.

Electron Carriers Involved in the Electron Transport Chain

Figure 7.13 Electron carriers in the electron transport chain.

Mitochondrial Shuttles

Two important shuttles that transport metabolites from the mitochondrial matrix to the cytosol

• Glycerophosphate shuttle
• Aspartate-malate shuttle

Mitochondrial Shuttles

Figure 7.14 Mitochondrial shuttles.

Complex I

$NADH$ dehydrogenase
Site of $NADH$ oxidation
Pumps 4 $H^{+}$ out of the mitochondrial matrix.
Electrons are transferred to ubiquinone.

Electron Transport in Complex I

Figure 7.16 Redox states of ubiquinone and FAD.

Complex II

Succinate dehydrogenase
Generates $FAD{H}_2$ and ubiquinone
No protons are pumped out of the matrix.
Contains two transmembrane and two hydrophilic domains.
Contains iron-sulfur centers.
Electrons are transferred to Complex III

Figure 7.17A Complex II.

Mechanism for Electron Transport in Complex II

Figure 7.18 Two plausible mechanisms for succinate dehydrogenase.

Complex III

Ubiquinone/cytochrome c reductase
Two ubiquinone binding sites
Heme is involved
Oxidation of one molecule of ubiquinone forms two molecules of reduced cytochrome c
Pumps 4 $H^{+}$ out of the mitochondrial matrix

Figure 7.19A Complex III.

Q Pool Defined

Combination of oxidized and reduced forms of ubiquinone found in the mitochondrial membrane

Q Cycle and Electron Transport

Figure 7.20 Q cycle and electron transport.

Cytochrome c

A soluble electron carrier
Receives electrons from Complex III
Heme protein that carries one electron in the heme group
Heme is buried in the core of the protein, but is reduced through quantum mechanics

Figure 7.21 Cytochrome c.

Complex IV

Cytochrome c oxidase
Takes 4 $e^{-}$ from cytochrome c
Pumps 2 $H^{+}$ out of the mitochondrial matrix
Reduces $O_2$ to $H_{2}O$
Uses four different electron carriers

Figure 7.22A Complex IV.

Mechanism for Electron Transport in Complex IV

Figure 7.23 Mechanism of water formation.
Oxygen atom acquires proton from tyrosine, which generates a tyrosyl radical.

Respirasome Defined

Respirasome is an aggregated supercomplex containing Complexes I, III, and IV.

• Other aggregated complexes include the replisome, transcriptional complex, and signal transduction complexes.
  Assists with substrate channeling

Figure 7.24 Respirasome.

Poisons That Inhibit the Electron Transport Chain

Structures of Poisons to the Electron Transport Chain

Figure 7.25 Poisons of electron transport.

Uncouplers and the Electron Transport Chain

Uncouplers interfere with electron transport.
Heat is generated instead of ATP.
DNP is an example of an uncoupler, as is UCP-1 (used by babies and hibernating animals)

Section 7.3 Learning Objective

Describe how the cell uses the electrochemical gradient produced in electron transport to synthesize ATP.

ATP Synthase

Also known as $F_o$/$F_1$ ATPase
Contains multiple subunits in both complexes
Enzyme mainly responsible for ATP production
Multimeric enzyme that uses electrochemical energy from the proton gradient to produce ATP from ADP and $P_i$

Structure of ATP Synthase

Figure 7.26A Reactions of ATP synthase.

Mechanism of ATP Synthase

Protons move between a and b subunits of $F_o$ complex.
Ring of c subunit rotates.
γ and ε subunits to also move through the hexamer of α and β subunits.
Conformational change occurs.
ATP is formed by binding of ADP and $P_i$.
Open, loose, and tight conformations

ATP Synthase Rotation

Figure 7.26C Reactions of ATP synthase.

Evidence of ATP Synthase Rotation

Figure 7.27 Rotation of the ATP synthase can be demonstrated in the laboratory.

Proton Motive Force Defined

Proton motive force is the electrochemical potential derived from the uneven distribution of electrons across the inner mitochondrial matrix.
Separated into a chemical and electric potential
ΔG = −2.303 RT ΔpH + nℱ ΔΨm

Chemical Potential Defined

Chemical potential is the higher concentration of protons on the outside of the membrane than on the inside.

Electric Potential Defined

Electric potential is the added positive charge that accumulates on the outside of the membrane.
Measured in V or mV
Voltage drop across the membrane

Gibbs Free Energy Correlation to Membrane Potential

Membrane potential (ΔΨm ) = Ψin
Ψin = voltage inside the membrane
Ψin = voltage outside the membrane
ΔG = nℱΔΨm
ℱ (F, Faraday’s constant) = 96.48 kJ/V · mol

ATP: ADP Translocase Defined

ATP: ADP translocase is an enzyme that transports ATP from the mitochondria in exchange for ADP
Transmembrane domain is an integral membrane protein spanning six α helices
Conformational change occurs

ATPases

Use ATP hydrolysis to perform their proscribed functions in reverse
Five different types

• F-type
• A-type
• V-type
• P-type
• E-type

ATPase Categories

Structures of Common ATPase Inhibitors