🔬Biological Chemistry I Unit 8 – Citric Acid Cycle & Oxidative Phosphorylation

Key Concepts and Overview

• Citric acid cycle (CAC), also known as tricarboxylic acid (TCA) cycle or Krebs cycle, is a central metabolic pathway for energy production
• Occurs in the mitochondrial matrix following glycolysis and oxidative decarboxylation of pyruvate
• Oxidative phosphorylation (OXPHOS) is the process of creating ATP using energy from redox reactions in the electron transport chain (ETC)
• The ETC consists of protein complexes I-IV embedded in the inner mitochondrial membrane that shuttle electrons from NADH and FADH2 to oxygen
• Proton gradient generated by ETC powers ATP synthase (complex V) to produce ATP from ADP and inorganic phosphate (Pi)
• CAC and OXPHOS are tightly regulated by substrate availability, energy demands, and allosteric effectors
• Dysfunction in CAC enzymes or OXPHOS complexes can lead to metabolic disorders and neurodegenerative diseases (Parkinson's, Alzheimer's)

Cellular Respiration Context

• Cellular respiration is the process of breaking down organic molecules to release energy in the form of ATP
• Consists of four stages: glycolysis (cytosol), pyruvate oxidation (mitochondria), citric acid cycle (mitochondrial matrix), and oxidative phosphorylation (inner mitochondrial membrane)
• Glycolysis converts glucose to pyruvate, generating 2 ATP and 2 NADH
• Pyruvate is transported into mitochondria and oxidized to acetyl-CoA by pyruvate dehydrogenase complex, releasing CO2 and NADH
• Acetyl-CoA enters the citric acid cycle for further oxidation
  • If oxygen is unavailable, pyruvate is reduced to lactate (anaerobic glycolysis) or ethanol (fermentation)
• NADH and FADH2 produced in the citric acid cycle are utilized in the ETC for oxidative phosphorylation

Citric Acid Cycle Breakdown

• The citric acid cycle is a series of eight enzymatic reactions that oxidize acetyl-CoA to CO2
• Acetyl-CoA condenses with oxaloacetate to form citrate, catalyzed by citrate synthase
• Citrate is isomerized to isocitrate by aconitase
• Isocitrate is oxidized and decarboxylated to α-ketoglutarate by isocitrate dehydrogenase, generating NADH and CO2
• α-Ketoglutarate is oxidized and decarboxylated to succinyl-CoA by α-ketoglutarate dehydrogenase complex, generating NADH and CO2
  • This complex is similar to pyruvate dehydrogenase complex and requires thiamine pyrophosphate (TPP), lipoic acid, FAD, NAD+, and CoA
• Succinyl-CoA is converted to succinate by succinyl-CoA synthetase, generating GTP (or ATP) and CoA
• Succinate is oxidized to fumarate by succinate dehydrogenase, the only membrane-bound enzyme in the cycle, reducing FAD to FADH2
• Fumarate is hydrated to malate by fumarase
• Malate is oxidized to oxaloacetate by malate dehydrogenase, regenerating NAD+ from NADH

Oxidative Phosphorylation Explained

• OXPHOS couples the redox reactions of the ETC with the phosphorylation of ADP to ATP
• Complex I (NADH dehydrogenase) oxidizes NADH, reducing ubiquinone (Q) to ubiquinol (QH2) and pumping protons into the intermembrane space
• Complex II (succinate dehydrogenase) oxidizes succinate to fumarate, reducing FAD to FADH2, which then reduces Q to QH2
• Complex III (cytochrome bc1 complex) oxidizes QH2 and reduces cytochrome c, pumping protons into the intermembrane space
  • Operates through the Q cycle, which involves a two-step reduction of Q at two different sites (Qo and Qi)
• Complex IV (cytochrome c oxidase) oxidizes cytochrome c and reduces oxygen to water, pumping protons into the intermembrane space
• The proton gradient generated by complexes I, III, and IV is used by ATP synthase to drive ATP production
  • Protons flow down their concentration gradient through the Fo subunit, causing rotation of the c-ring
  • Rotation of the c-ring is coupled to conformational changes in the F1 subunit, catalyzing ATP synthesis from ADP and Pi

Enzymes and Coenzymes Involved

• The citric acid cycle and oxidative phosphorylation involve several enzymes and coenzymes
• Citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase complex, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase are the eight enzymes of the citric acid cycle
• Pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA, linking glycolysis to the citric acid cycle
• Coenzymes NAD+ and FAD are reduced to NADH and FADH2, respectively, during the citric acid cycle and serve as electron donors for the ETC
• Thiamine pyrophosphate (TPP), lipoic acid, and coenzyme A (CoA) are essential cofactors for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes
• Ubiquinone (coenzyme Q10) and cytochrome c are mobile electron carriers in the ETC
• Heme and copper centers in cytochrome c oxidase are crucial for electron transfer and oxygen reduction
• Magnesium ions (Mg2+) are required for ATP synthase activity

Energy Production and ATP Synthesis

• The citric acid cycle generates one ATP (or GTP) per turn through substrate-level phosphorylation catalyzed by succinyl-CoA synthetase
• Each NADH produced in the citric acid cycle yields ~2.5 ATP, while each FADH2 yields ~1.5 ATP through oxidative phosphorylation
• One glucose molecule can generate up to 38 ATP: 2 from glycolysis, 2 from the citric acid cycle, and 34 from oxidative phosphorylation
  • This value is a theoretical maximum; the actual yield is lower due to proton leakage and the cost of transporting ATP out of the mitochondria
• ATP synthase is a rotary engine that couples proton flow to ATP synthesis
  • The proton-motive force drives rotation of the c-ring in the Fo subunit, which is coupled to conformational changes in the F1 subunit
  • The F1 subunit contains three catalytic sites that cycle through open, loose, and tight states, binding ADP and Pi, forming ATP, and releasing ATP
• The P/O ratio is the number of ATP molecules produced per pair of electrons transferred in the ETC (~2.5 for NADH and ~1.5 for FADH2)

Regulation and Control Mechanisms

• The citric acid cycle and oxidative phosphorylation are tightly regulated to maintain energy balance and prevent oxidative stress
• Citrate synthase is inhibited by high levels of ATP and NADH, which indicate a high energy state
• Isocitrate dehydrogenase is allosterically stimulated by ADP and inhibited by ATP and NADH
• α-Ketoglutarate dehydrogenase complex is inhibited by high levels of succinyl-CoA and NADH
• Pyruvate dehydrogenase complex is inhibited by acetyl-CoA and NADH and activated by calcium ions (Ca2+)
• The electron transport chain is regulated by the availability of substrates (NADH and FADH2) and the proton gradient
  • A high proton gradient slows electron transport, while a low gradient stimulates it
• ATP synthase is inhibited by a high ATP/ADP ratio, which indicates a high energy state
• Uncoupling proteins (UCPs) can dissipate the proton gradient without ATP synthesis, generating heat and reducing oxidative stress

Clinical and Real-World Applications

• Mitochondrial disorders can arise from mutations in genes encoding citric acid cycle enzymes or oxidative phosphorylation complexes
  • Leigh syndrome is caused by defects in pyruvate dehydrogenase complex, complex I, or complex IV, leading to neurodegeneration and lactic acidosis
  • MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is caused by mutations in mitochondrial tRNA genes, impairing protein synthesis
• Neurodegenerative diseases such as Parkinson's and Alzheimer's have been linked to mitochondrial dysfunction and oxidative stress
  • Parkinson's disease is associated with impaired complex I activity and accumulation of α-synuclein, leading to dopaminergic neuron loss
  • Alzheimer's disease is characterized by amyloid-β and tau protein aggregation, which can disrupt mitochondrial function and energy production
• Metabolic disorders such as obesity and type 2 diabetes can affect mitochondrial function and energy metabolism
  • Insulin resistance can impair glucose uptake and oxidation, leading to reduced ATP production and increased oxidative stress
• Mitochondrial-targeted antioxidants (MitoQ, SS-31) and metabolic modulators (metformin, resveratrol) are being investigated as potential therapies for mitochondrial dysfunction and related disorders
• Exercise and caloric restriction have been shown to improve mitochondrial function, increase ATP production, and reduce oxidative stress, promoting overall health and longevity