11.4 Biochemical Energetics

ATP and other high-energy phosphates

Cells do not store electricity. They store phosphoryl-transfer potential. Adenosine triphosphate (ATP) has two phosphoanhydride bonds. Under biochemical standard conditions, hydrolysis of ATP to ADP + Pi has ΔG°′ ≈ −30.5 kJ/mol (−7.3 kcal/mol). In the crowded, Mg2+-chelated cytosol the actual ΔG is even more negative (often near −50 kJ/mol), which is why ATP hydrolysis can drive otherwise endergonic steps. The useful equation is ΔG = ΔG°′ + RT ln Q: mass action, not the standard number alone, decides whether a reaction runs forward in a hepatocyte.

Not every phosphate is “high energy.” Compare standard free energies of hydrolysis:

Substrate-level phosphorylation is ATP (or GTP) made by transferring a phosphate from a high-energy substrate without the electron-transport chain: phosphoglycerate kinase, pyruvate kinase, and succinyl-CoA synthetase (GTP). Oxidative phosphorylation is ATP made by ATP synthase using the proton-motive force. Creatine kinase buffers muscle and nerve ATP: creatine phosphate + ADP ⇌ creatine + ATP. That near-equilibrium pool is why a short isometric effort can continue after free ATP would have been exhausted.

Adenylate kinase (2 ADP ⇌ ATP + AMP) makes AMP a sensitive signal of energy charge. High AMP activates AMP-activated protein kinase (AMPK) and phosphofructokinase-1, matching glycolysis to energy need. Energy charge (Atkinson) is a weighted ATP/ADP/AMP ratio; you do not need the formula, but you do need the idea that ATP-generating pathways turn on when charge falls.

Quick Answer: NADH donates to Complex I (~2.5 ATP). FADH2 donates to Complex II (~1.5 ATP). Complexes I, III, and IV pump protons; II does not. Cyanide blocks IV. Uncouplers leak protons (O2 and heat up, ATP down). Oligomycin blocks ATP synthase (O2 down unless you add an uncoupler).

Redox pairs: NADH and FADH2

Oxidation is electron loss; reduction is electron gain. Catabolism is a controlled burn: carbons in fuels are oxidized to CO2 while NAD+ and FAD are reduced. NAD+ accepts a hydride (two electrons and one proton); the second proton is released to solvent. NAD-linked dehydrogenases are usually soluble (malate dehydrogenase, isocitrate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase). FAD (and FMN) can accept one electron at a time, which is why FAD prosthetic groups sit in membrane enzymes that must talk to iron-sulfur clusters and ubiquinone (succinate dehydrogenase, acyl-CoA dehydrogenase).

Electrons flow toward more positive standard reduction potential (E°′). NADH has E°′ ≈ −0.32 V; the ½ O2 / H2O couple is +0.82 V. The large ΔE is the thermodynamic permission for the electron-transport chain. Approximate relationship: ΔG°′ = −n F ΔE°′. Two electrons from NADH to oxygen release enough free energy to pump protons and still make ATP; the leftover energy is heat even in coupled mitochondria.

Modern P/O ratios (ATP per atom of oxygen, equivalently per 2 electrons) are about 2.5 for NADH and 1.5 for FADH2. Older textbooks used 3 and 2. Either scale is acceptable if you are internally consistent; Part I items more often ask relative yield (NADH > FADH2 because Complex I pumps) than a single integer. Complete oxidation of glucose is classically 30–32 ATP (or 36–38 on the old scale), depending on whether the malate-aspartate shuttle (NADH-equivalent) or the glycerol-3-phosphate shuttle (FADH2-equivalent) reoxidizes cytosolic NADH from glycolysis.

Citric acid cycle

The citric acid cycle (Krebs, TCA) in the mitochondrial matrix oxidizes the acetyl group of acetyl-CoA. Condensation of acetyl-CoA with oxaloacetate by citrate synthase yields citrate. Aconitase isomerizes to isocitrate. Isocitrate dehydrogenase performs the first oxidative decarboxylation (NADH + CO2) to alpha-ketoglutarate. Alpha-ketoglutarate dehydrogenase (the PDH-like complex: TPP, lipoamide, CoA, FAD, NAD+) performs the second oxidative decarboxylation to succinyl-CoA. Succinyl-CoA synthetase makes GTP (substrate-level). Succinate dehydrogenase (Complex II) oxidizes succinate to fumarate (FADH2). Fumarase adds water; malate dehydrogenase makes oxaloacetate (NADH), and the cycle turns.

Per acetyl-CoA: 3 NADH + 1 FADH2 + 1 GTP. Using modern P/O: 3 × 2.5 + 1.5 + 1 = 10 ATP equivalents. Pyruvate dehydrogenase (not a TCA enzyme, but the gate) adds another NADH per pyruvate. Anaplerosis refills the cycle: pyruvate carboxylase (biotin; activated by acetyl-CoA) is the chief liver route to oxaloacetate. Cataplerosis drains intermediates (citrate export for fatty acid synthesis).

Regulation hits the irreversible steps. Isocitrate dehydrogenase: activated by ADP and Ca2+, inhibited by NADH and ATP. Alpha-ketoglutarate dehydrogenase: inhibited by NADH, succinyl-CoA, and ATP; activated by Ca2+ (working muscle). Citrate synthase is limited by oxaloacetate availability and, in some texts, by citrate and NADH. A high NADH/NAD+ ratio means the chain is backed up; TCA dehydrogenases stop so you do not generate reducing equivalents you cannot reoxidize.

Electron-transport chain, proton-motive force, and oxidative phosphorylation

The inner mitochondrial membrane is the working wall. Complex I (NADH:ubiquinone oxidoreductase) oxidizes matrix NADH via FMN and iron-sulfur centers and reduces ubiquinone (coenzyme Q) to ubiquinol. It pumps four protons. Inhibitors: rotenone, piericidin A, amytal (amobarbital), MPP+ (the parkinsonism toxin from MPTP). Succinate can still feed the chain distal to I.

Complex II (succinate dehydrogenase) oxidizes succinate, reduces Q, and pumps zero protons. That is why FADH2-derived electrons yield less ATP. Malonate is a competitive inhibitor of Complex II.

Complex III (cytochrome bc1, ubiquinol-cytochrome c oxidoreductase) runs the Q cycle, reduces cytochrome c (a mobile heme protein in the intermembrane space), and pumps four protons per 2 electrons. Antimycin A blocks the Qi site and stops oxidation of both NADH and succinate.

Complex IV (cytochrome c oxidase, a/a3) transfers electrons from cytochrome c onto molecular oxygen, making water. Copper centers (CuA, CuB) and heme a3 form the O2-reduction site. It pumps two protons per 2 electrons. Inhibitors: cyanide, azide, carbon monoxide. Cyanide binds ferric heme a3; CO binds ferrous heme a3 and also hemoglobin. Cyanide leaves venous blood looking oxygenated because extraction stops; lactic acidosis is severe. Antidote chemistry: nitrites generate methemoglobin as a cyanide sink, thiosulfate feeds rhodanese to make thiocyanate, and hydroxocobalamin directly chelates cyanide.

Ubiquinone is the lipid-soluble collector from I, II, and several other FAD dehydrogenases (including ETF from beta-oxidation). Cytochrome c is the water-soluble collector between III and IV. Blocking III or IV therefore darkens both NADH and FADH2 oxidation; blocking I leaves succinate as a bypass.

Proton-motive force (Δp) has two terms: membrane potential (Δψ), negative inside the matrix, and pH gradient (ΔpH), alkaline matrix. Protons re-enter through ATP synthase (F0F1, Complex V). F0 is the membrane proton pore; F1 is the matrix catalytic knob. Rotation of the gamma subunit drives the binding-change mechanism: ADP + Pi → ATP. Rough proton arithmetic: Complex I+III+IV pump about 10 H+ per NADH; about 4 H+ are needed per ATP when you count synthase plus the cost of phosphate import and adenine-nucleotide exchange — hence ~2.5 ATP/NADH. FADH2 skips Complex I, so about 6 H+ and ~1.5 ATP.

Adenine nucleotide translocase (ANT) exchanges matrix ATP4− for cytosolic ADP3− (electrogenic, driven by Δψ). Phosphate carrier brings Pi with a proton. Atractyloside and bongkrekic acid inhibit ANT: mitochondria cannot get ADP in or ATP out, so synthase stalls even if the chain is intact.

Inhibitors versus uncouplers

This table is the highest-yield energetics object on Part I. An ETC inhibitor blocks electron flow: NADH stays reduced, oxygen consumption falls, the proton gradient collapses after existing protons leak, and ATP falls. An ATP synthase inhibitor (oligomycin) blocks proton re-entry through F0: the gradient becomes maximal, electron flow stops because protons have nowhere to go, oxygen consumption falls, and ATP falls. An uncoupler is a protonophore (or a protein pore) that lets protons re-enter without synthase: the gradient collapses, electron flow and oxygen consumption rise (respiration is released from respiratory control), energy becomes heat, and ATP falls.

2,4-Dinitrophenol was an infamous weight-loss toxin: uncontrolled heat, hyperthermia, and death. Aspirin overdose uncouples at high dose and produces an initial respiratory alkalosis plus later lactic acidosis — a physiology crossover. UCP1 in brown adipose tissue is the physiologic uncoupler of the neonate, activated via beta-3 adrenergic lipolysis and free fatty acids.

The classic isolated-mitochondria experiment: oxidize NADH, measure O2. Add oligomycin → O2 stops. Add DNP → O2 resumes, ATP does not. If the first poison had been cyanide, DNP could not resume O2 use, because electrons cannot reach oxygen. That single branching experiment distinguishes synthase block from chain block from uncoupling.

Respiratory control is the dependence of ETC flux on ADP availability: no ADP, synthase stops, gradient maxes, NADH cannot be oxidized, TCA stops. Uncouplers destroy respiratory control. Ischemia is the clinical inverse of cyanide: no oxygen acceptor, same NADH-reduced, ATP-depleted ending, with Na+/K+-ATPase failure and cell swelling — the biochemistry of reversible then irreversible injury.

Keep Complex numbers, pump-versus-no-pump, and the oligomycin/DNP split memorized as a unit. Then TCA yields and ATP hydrolysis numbers have a place to land.