The Mitochondrial Respiratory Chain

• The outer membrane is readily permeable to small molecules and ions. Transport occurs through porins.
• The inner membrane is impermeable to most small molecules and ions, so transport requires specific transporters.
• The outer membrane is freely permeable to small molecules and ions.
• The inner membrane:

  • Impermeable to most small molecules and ions, including H+
  • Contains respiratory electron carriers (complexes I to IV), ADP to ATP translocase, ATP synthase (F0F1), and other membrane transporters
• The matrix contains:

  • Pyruvate dehydrogenase complex
  • Citric acid cycle enzymes
  • Fatty acid beta-oxidation enzymes
  • Amino acids oxidation enzymes
  • DNA, ribosomes
  • Many other enzymes
  • ATP, ADP, Pi, Mg2+, Ca2+, K+
  • Many soluble metabolic intermediates.
• The inner mitochondrial membrane segregates the intermediates and enzymes of cytosolic and matric metabolic pathways.
• Which of these dehydrogenase enzymes is not found in the mitochondrial matrix?

  • Malate dehydrogenase
  • Glutamate dehydrogenase
  • Acyl CoA dehydrogenase
  • Lactate dehydrogenase

  Lactate dehydrogenase. The mitochondrial matric contains enzymes of the citric acid cycle (malate dehydrogenase), the beta-oxidation pathway (acyl CoA dehydrogenase) and amino acid oxidation (glutamate dehydrogenase). The enzymes of glycolysis and fermentation (lactate dehydrogenase) are located in the cytosol.
• Cristae are convolutions in the inner membrane of the mitochondrion.

  • Mitochondria of cells with high metabolic activity have more cristae.
• During cell growth and division, mitochondria divide by fission.
• Stressful conditions can trigger:

  • Mitochondrial fission
  • Mitophagy, the breakdown of mitochondria and recycling amino acids, nucleotides and lipids
• As stress is relieved, small mitochondria fuse to form long, thin, tubular organelles.
• The respiratory chain is a series of electron carriers.
• Dehydrogenases collect electrons from catabolic pathways and funnel them into universal electron acceptors

  • Nicotinamide nucleotides (NAD+ or NADP+)
  • Flavin nucleotides (FMN or FAD)
• Nicotinamide nucleotide linked dehydrogenases catalyze reversible reactions of these general types:

  • Reduced substrate + NAD+ --> oxidized substrate + NADH + H+
  • Reduced substrate + NADP+ --> oxidized substrate + NADPH + H+
• Two hydrogen atoms are removed from the substrates:

  • One is transferred as a hydride ion to NAD(P)+
  • One is released as H+ in the medium
• NAD+ linked reactions:

  • alpha-ketoglutarate + CoA + NAD+ --> succinyl CoA + CO2 + NADH + H+ (mitochondria)
  • L-malate + NAD+ --> oxaloacetate + NADH + H+ (mitochondria and cytosol)
  • Pyruvate + CoA + NAD+ --> acetyl CoA + CO2 + NADH + H+ (mitochondria)
  • Glyceraldehyde 3-phosphate + Pi + NAD+ --> 1,3-bisphosphoglycerate + NADH + H+ (cytosol)
  • Lactate + NAD+ --> pyruvate + NADH + H+ (cytosol)
  • Beta-hydroxyacyl CoA + NAD+ --> beta-ketoacyl CoA + NADH + H+ (mitochondria)
• NADP+ linked reactions:

  • Glucose 6-phosphate + NADP+ --> 6-phosphogluconate + NADPH + H+ (cytosol)
  • L-malate + NADP+ --> pyruvate + CO2 + NADPH + H+ (cytosol)
• NAD+ or NADP+ linked reactions:

  • L-glutamate + H2O + NAD(P)+ --> alpha-ketoglutarate + NH4+ + NAD(P)H (mitochondria)
  • Isocitrate + NAD(P)+ --> alpha-ketoglutarate + CO2 + NAD(P)H + H+ (mitochondria and cytosol)
• What enzyme would not be expected to contribute electron carriers to oxidative phosphorylation?

  • Alcohol dehydrogenase
  • Malate dehydrogenase
  • Succinate dehydrogenase
  • Glucose 6-phosphate dehydrogenase
  • Glyceraldehyde 3-phosphate dehydrogenase

  Glucose 6-phosphate dehydrogenase. It produces NADPH, which does not contribute electrons to the respiratory chain.
• Flavoproteins contain a very tightly, sometimes covalently, bound flavin nucleotide (FMN or FAD)
• The oxidized flavin nucleotide can accept either:

  • One electron, yielding the semiquinone form
  • Two electrons, yielding FADH2 or FMNH2
• Electron transfer occurs because the flavoprotein has a higher Edegree than the compound oxidized

  • E degree of a flavin nucleotide depends on the protein with which it is associated
• Three types of electron transfers occur in oxidative phosphorylation:

  • Direct transfer of electrons
  • Transfer as a hydrogen atom (H+ + electon)
  • Transfer as a hydride ion
• Reducing equivalent is a single electron equivalent transferred in an oxidation reduction reaction/
• What ion, atom, or molecule constitutes one reducing equivalent?

  • Proton, H+
  • Hydrogen atom (H+ + electron)
  • Hydride ion
  • NADH

  Hydrogen atom (H+ + electron). The term reducing equivalent is used to designate a single electron equivalent transferred in an oxidation reduction reaction. A proton cannot act as a reducing equivalent because it has no electrons. A hydrogen atom acts as one reducing equivalent. A hydride ion or NADH molecule acts as two reducing equivalents.
• Five types of electron carrying molecules:

  • NAD
  • Flavoproteins
  • Ubiquinone (coenzyme Q or Q)
  • Cytochromes
  • Iron sulfur proteins
• Ubiquinone is a lipid soluble benzoquinone with a long isoprenoid side chain

  • Can accept one or two electrons
  • Freely diffusible within the inner mitochondrial membrane
  • Plays a central role in coupling electron flow to proton movement
• Cytochromes are proteins with characteristic strong absorption of visible light due to their iron containing prosthetic groups

  • One electron carriers
  • Three classes in the mitochondria, a, b and c
  • Hemes a and b are not covalently bound to associated proteins
  • c is covalently attached through Cys residues
• Which statement is false about the cytochrome electron carriers?

  • Cytochromes a, b, and c are distinguished by their differences in their light absorption spectra.
  • Soluble cytochrome c associates with the outer surface of the inner membrane through electrostatic interactions
  • The heme of cytochrome c is tightly, but not covalently bound to its associated protein
  • Cytochromes a and b are integral proteins of the inner mitochondrial membrane.

  The heme of cytochrome c is tightly, but not covalently bound, to its associated protein.
• Cytochrome c has an absorption peak at 400nm.
• Cytochrome b has an absorption peak at 500 nm.
• Cytochrome a has an absorption peak at 550 nm.
• Iron sulfur proteins that contain iron in association with inorganic sulfur atoms and/or with the sulfur atoms of Cys residues in the protein

  • Participate in one electron transfers
• Rieske iron sulfur protein are proteins in which one iron atom is coordinated with two His residues.
• Which compound is not an electron carrier involved in the respiratory chain?

  • NADH
  • Iron sulfur proteins
  • Cytochromes
  • Coenzyme A

  Coenzyme A. It serves multiple metabolic functions in both anabolic and catabolic pathways; however, it does not act as an electron carrier in the respiratory chain.
• Determining the sequence of electron carrier can be done by:

  • determine the E'degree of the individual electron carriers
  • reduce the entire chain by omitting oxygen and then measure the oxidation rate of each electron carrier when oxygen is reintroduced.
  • Measure the effects of inhibitors of electron transfer on the oxidation state of each carrier
• Electrons tend to flow spontaneously from carriers of lower E'degree to carriers of higher E'degree
• The order to carriers is:

  • NADH --> Q --> cytochrome b --> cytochrome c1 --> cytochrome c --> cytochrome a --> cytochrome a3 --> oxygen
  • This order has been confirmed by all three approaches
• Four unique electron carrier complexes catalyze electron transfer through a portion of the chain:

  • Complex I (NADH to ubiquinone)
  • Complex II (succinate to ubiquinone)
  • Complex III (ubiquinone to cytochrome c)
  • Complex IV (cytochrome c to oxygen)
• Which electron carrier complex in the respiratory chain oxides ubiquinone?

  • Complex I
  • Complex II
  • Complex III
  • Complex IV

  Complex III. It carries electrons from reduced ubiquinone to cytochrome c.