Biochemistry of Electron Transport Chain

Oxidative Phosphorylation

• Oxidative phosphorylation is a central role in eukaryotic aerobic metabolism.
• ATP production isn't the only function of mitochondria.
• Mitochondria host the citric acid cycle and fatty acid β-oxidation pathways.
• Mitochondria are involved in amino acid oxidation, thermogenesis, steroid synthesis, and apoptosis.
• Mitochondrial discovery has stimulated current research on mitochondrial biochemistry.
• Mitochondria trace their evolutionary origin to bacteria.
• An endosymbiotic relationship existed between bacteria and primitive eukaryotes over 1.45 billion years ago.
• Mitochondria are ubiquitous in modern eukaryotes.
• Their bacterial origin is evident in almost every aspect of their structure and function.
• Electrons flow from electron donors to a final electron acceptor with a large reduction potential in a chain of membrane-bound carriers.
• Molecular oxygen (O2) is the final acceptor.
• The appearance of oxygen in the atmosphere approximately 2.3 billion years ago, and its use in living systems through oxidative phosphorylation, made complex life forms possible.
• The free energy from electron flow is coupled with the transport of protons across a proton-impermeable membrane.
• Fuel oxidation free energy is thus conserved as a transmembrane electrochemical potential.
• Transmembrane proton flow through specific protein channels provides the free energy for ATP synthesis.
• The process is catalyzed by a membrane protein complex (ATP synthase).
• This complex couples proton flow to phosphorylation of ADP.
• The chemiosmotic theory describes the relationship between the transmembrane difference and ATP synthesis.
• The inner mitochondrial membrane is impermeable to most small molecules and ions.
• Transport across this membrane requires specific transporters; porins allow small molecules to pass through the outer membrane.
• The mitochondrial matrix contains the pyruvate dehydrogenase complex, citric acid cycle enzymes, fatty acid β-oxidation pathway enzymes, and amino acid oxidation pathways enzymes.
• The inner mitochondrial membrane segregates cytosolic and matrix metabolic pathways intermediates and enzymes.
• The order of carriers in the electron transport chain is NADH → Q → cytochrome b → cytochrome c₁ → cytochrome c → cytochrome a → cytochrome a3 → O2.
• This order has been confirmed by three approaches.
• Ubiquinone (Coenzyme Q) and cytochromes are electron-carrying molecules in the respiratory chain.
• Flavoproteins and iron-sulfur proteins are also involved.
• Cytochromes a, b, and c are distinguished by their light-absorption spectra.
• Cytochrome c is loosely bound to its associated protein.
• Cytochromes a and b are integral proteins of the inner mitochondrial membrane.
• Iron-sulfur proteins contain iron in association with inorganic sulfur atoms.
• They participate in one-electron transfers.
• Complex I (NADH dehydrogenase), Complex II (succinate dehydrogenase), Complex III (ubiquinone-cytochrome c oxidoreductase), and Complex IV (cytochrome oxidase) catalyze electron transfer in separate but linked phases of respiration.
• Flavoproteins and iron-sulfur proteins are important components in complex electron transfer reactions.
• Ubiquinone (coenzyme Q) readily diffuses within the inner mitochondrial membrane and plays a central role in coupling electron flow to proton movement.
• Cytochromes are proteins with characteristic strong absorption of visible light due to iron-containing heme prosthetic groups.
• They are one-electron carriers in the electron transport chain.
• The mitochondrial matrix contains enzymes of the citric acid cycle (malate dehydrogenase), the β-oxidation pathway (acyl-CoA dehydrogenase), and amino acid oxidation (glutamate dehydrogenase).
• Glycolysis and fermentation enzymes (lactate dehydrogenase) are located in the cytosol.
• Cristae are convolutions in the inner membrane of the mitochondrion.
• Mitochondria in cells with high metabolic activity have more cristae.
• During cell growth and division, mitochondria divide by fission.
• Stressful conditions can trigger mitochondrial fission.
• Mitophagy is the breakdown of mitochondria and amino acid, nucleotide, and lipid recycling.
• Small mitochondria fuse to form long, thin, tubular organelles when stress is relieved.
• The respiratory chain consists of a series of electron carriers.
• Dehydrogenases collect electrons and funnel them into universal electron acceptors NAD+ or NADP+ and flavin nucleotides (FMN or FAD).
• Nicotinamide nucleotide-linked dehydrogenases catalyze reversible reactions of the NAD+ or NADP+ types.
• Two hydrogen atoms are removed from the reduced substrates.
• One is transferred as a hydride ion (:H-) to NAD(P)+.
• One is released as H+ into the medium.
• Important reactions are catalyzed by NAD(P)+-linked dehydrogenases (Table 19-1).
• Oxidative phosphorylation requires electron flow from electron donors to a final electron acceptor, typically molecular oxygen (O2).
• The flow of electrons through Complexes I, II, III, and IV results in the pumping of protons across the inner mitochondrial membrane, creating a proton gradient across the membrane.
• The electron-transport chain of the respiratory chain generates ATP by creating a proton-motive force.
• The proton-motive force provides energy for ATP synthesis from ADP and Pi.
• The chemiosmotic model describes the coupling of ATP synthesis to an electrochemical proton gradient.
• The proton-motive force is composed of chemical and electrical potential energy, resulting from the difference in [H+] gradient.
• 2 NADH + 2H+ + O2 → 2NAD+ + 2H2O
• The mass-action ratio (ATP)/([ADP][Pi]) is a measure of a cell's energy status.
• A rise in ADP concentration leads to an increased rate of respiration.
• Inhibitory proteins such as IF1 can prevent ATP hydrolysis during hypoxic conditions.
• Chemical uncouplers such as DNP and FCCP dissipate the proton gradient.
• In the absence of an oxidizable substrate, the proton-motive force alone drives ATP synthesis.
• Species differences exist in the number of c subunits in the F₀ complex, and this impacts the P/O ratio.
• Mitochondrial ATP synthase F₁ has an α3β3γδ subunits composition. Each ẞ subunit can assume three different conformations: β-ATP (tight binding), β-ADP (loose binding), and β-empty (very loose binding).
• The Fo complex has ab2cn composition (n from 8–17). c ring is an arrangement of c subunits into two concentric circles.
• Proton translocation causes a rotation of F₀ and Y, causing conformational changes within αβ pairs, and promotes ADP + Pi condensation to ATP.
• The y subunit rotates in one direction when FoF₁ synthesizes ATP, and in the opposite direction when hydrolyzing ATP.
• The glycerol 3-phosphate shuttle is necessary for oxidative phosphorylation in the brain.
• The malate-aspartate shuttle is necessary for oxidative phosphorylation in liver.
• Mitochondrial genomes are less efficient at repairing DNA damage in comparison to the nuclear genome and are primarily inherited from the female lineage.
• Mutations in mitochondrial DNA accumulate over an organism's lifetime and frequently cause disease with heteroplasmic cells having varying degrees of severity.
• The energy of electron transfer is efficiently conserved in a proton gradient. The change in standard reduction potential is 1.14 V.
• The standard free-energy change is -220 kJ/mol of NADH.
• Mitochondrial P-450 monooxygenases catalyze steroid hydroxylations, which occur in steroidogenic tissues.
• The cytochrome P-450 family catalyzes a series of hydroxylation reactions to synthesize steroid hormones.
• A flavoprotein and an iron-sulfur protein carry electrons from NADPH to the P-450 heme. All P-450 enzymes have a heme that interacts with O2, and a substrate binding site for specificity.
• The inner mitochondrial membrane is impermeable to NADH and NAD+. Cytosolic NADH must be transported into mitochondria.
• Specific shuttle systems carry reducing equivalents from cytosolic NADH into mitochondria.
• Mitochondrial DNA defects frequently occur due to reactive oxygen species (ROS) exposure, and mtDNA replication system having lower correction effectiveness compared to the nuclear system.
• Animals inherit their mitochondria essentially solely from the female parent.
•  The chemiosmotic mechanism likely evolved before the emergence of eukaryotes.
• Mitochondrial donation implies transplantation of an ovum's nuclear genes into an enucleated ovum. This procedure creates an ovum free of mutations that leads to a mitochondrial disease and raises ethical issues.

Thermogenesis

• Brown adipose tissue (BAT) is an adipose tissue in newborn mammals.
• It generates heat through fuel oxidation.
• Uncoupling protein 1 (UCP1) is a long-chain fatty acid/H+ symporter in BAT.
• It provides a path for protons to return to the matrix without passing through the FoF₁ complex.
• This action results in dissipation of oxidation energy as heat.
• Hydrolysis of phosphocreatine also releases heat in thermogenesis.
• Hibernating animals depend on uncoupled BAT mitochondria activity to generate heat during their long dormancy periods.

Steroid Synthesis

• Mitochondria are the site of steroid hormone production.
• Steroidogenic tissues (adrenal gland, gonads, liver, kidney) contain these reactions.
• Mitochondria contain cytochrome P-450 enzymes involved in steroid synthesis.
• The reactions catalyze a series of hydroxylations to synthesize steroid hormones from a sterol.
• These enzymes possess a critical heme group.

Apoptosis

• Apoptosis (programmed cell death) is a cellular process where individual cells die for the organism's benefit.
• It can be triggered during normal embryonic development, by external signals, or from internal events (DNA damage, viral infection, oxidative stress, etc.).
•  Mitochondria are central to apoptosis initiation.
• Permeability transition pore complex (PTPC) is a multisubunit membrane protein that increases membrane permeability.
• Apaf-1 (apoptosis protease activating factor-1) monomers interact with cytochrome c from mitochondria, resulting in the formation of an apoptosome.
•  Caspases are proteases that are crucial during the apoptosis process.

Mitochondrial Genes

• Thirteen mitochondrial proteins are encoded by the mitochondrial genome and synthesized within mitochondria.
• Approximately 1,200 mitochondrial proteins are encoded by nuclear genes and imported into mitochondria.
•  Mitochondrial genes are often associated with a high level of specific disease outcomes depending on the specific mutations and the organism.
• The human mitochondrial genome is circular rather than linear and is less efficient in repair compared to nuclear DNA.
• The mitochondrial genome is predominantly inherited from the maternal lineage.
• Mitochondrial encephalomyopathies represent a group of disorders that mainly affect the brain and skeletal muscle.
• Leber hereditary optic neuropathy (LHON) is a rare ailment associated with mutations within the mitochondrial Complex 1 (e.g. a single amino acid substitution.)
•  Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome results from a mutation within a tRNA gene.

Oxidative Phosphorylation Regulation

• Acceptor control of respiration is regulated by ADP, which is the major P₁ acceptor.
• The rate of O2 consumption increases with the availability of ADP.
• The acceptor control ratio is the maximal rate of ADP-induced O2 consumption divided by the basal rate in the absence of ADP; this ratio is at least 10 for some animal tissues.
• The mass-action ratio([ATP]/([ADP][P;])) is a crucial factor in regulating oxidative phosphorylation rates. Higher ADP levels correlate with more active oxidative phosphorylation, when ADP is depleted, so does the rate of oxidative phosphorylation.
• Oxidative phosphorylation is coordinated with the rates of electron transfer, the citric acid cycle, pyruvate oxidation, and glycolysis as cellular energy needs change.