GC550: Mitochondria Function and Structure: Oxidative Phosphorylation, ETC, Proton Motive Force generation and uncoupling

Mitochondrial Structure

• Smooth outer membrane and highly convoluted inner membrane, generating multiple distinct mitochondrial compartments: intermembrane space and mitochondrial matrix

• Cristae: intermembrane space subcompartment formed by inner membrane folds, connected to the boundary intermembrane space by cristae junctions

Overview of Mitochndrial Functions

• mediate apoptosis (cell life-death decisions)

• Cell signaling, epigenetic regulation

• Cellular Ca2+ signaling, which can impact many cytosolic/nuclear processes

• Make heme and generate urea

• Cellular Ca2+ homeostasis and Ca2+ signaling

Metabolite/Ion transport across OMM vs IMM

OMM

• contains large diameter pore-like channel VDAC → broad specificity for molecules of MW <5000, open and closed state may be subject to regulation

• relatively porous

  IMM

• not porous, tight

• contains transporters and channels for metabolite/ions (Inorganic phosphate, ATP, ADP, Pyruvate, di and tricarbxylic acids, amino acids, long chain acyl carnitine esters, Na+, K+, Ca2+)

• Free diffusion of O2 and CO2

• highly regulated

• We want this to establish the electrochemical gradient necessary to generate ATP

• A large extent of H+ transport across IMM is via protein complexes involved in ox phos

Oxidative Phosphorylation Overview

• coupling of substrate dilation and phosphorylation of ADP through a proton electrochemical gradient

ATP Hydrolysis vs ADP phosphorylation

• ATP hydrolysis is highly exergonic (releases energy) → energy captured from this reaction can be used to drive endergonic reactions (energy stored in ATP used to drive reactions)

• ADP phosphorylation/ATP synthesis is highly endergonic (unfavorable, input of energy)

Proton Pumps

• key to electrochemical energy conservation

• Oxidative phosphorylation relies on two types of proton pump with the same orientation located in the same membrane

  1. ATPase proton pump → energy of ATP hydrolysis can be used to drive electron transport upstream to reduce NAD+ and NADH (reverse of ox phos)

  2. Electron shuttle → driven by the free energy change of redox reactions

• Coupling of these pumps (redox-driven proton pump with an ATP-driven proton pump in the same membrane) permits reversal of the ATPase reaction driven by the free energy change of the redox reactions

Free Energy of Membrane Transport Reactions

• Uncharged molecular transport → driving force (free energy change) s determined by concentration gradient

• Charged molecular transport → driving force is determines by concentration gradient and by the electrochemical potential difference across the membrane

• Mitochondrial Proton Motive Force (∆p) → generated by [H+] → concentration gradient (pH) and electrical gradient (charge separation)

• Mainly composed of voltage change/charge separation

Generation of ∆p

• ETC is a chain of redox of reactions that generate free energy to pump protons

  1. Three complexes shuttle electrons → NADH/FADH2 reducing equivalents get oxidized which drives proton pumping in complex I (FADH2 oxidized by complex II but complex II does not pump protons)

  2. Electrons are shuttled by ubiquinone (Q) to complex III, which also drives H+ pumping

  3. Electrons are shuttled to cytochrome c and onto complex IV which also pumps protons

  4. O2 is final e- accepter and water is produced → Liberates free energy that drives proton pumping through complex IV

• Electrons enter at Complex I OR II (via NADH or FADH2)

• Coupled to CAC through succinate dehydrogenase to Q

Electron Carriers in the Mitochondrial Respiratory Chain

Mitochondrial ETC Redox Reactions

• different affinities creates directionality

Redox Potential in ETC

• redox potential increases as electrons flow down the respiratory chain to oxygen and can be directly related to the release of free energy

• Direction of e- flow depends on affinity of different e- carriers

• Affinity: expressed as a redox potential - the affinity of any pair of oxidized and reduced compounds (redox pair) for electrons

• NADH has poor affinity for e- → very good electron donor

• O2 has high affinity for e- → readily reduced, good electron acceptor

• Direction of electron flow is from compounds with lower redox potential to compounds with successively higher redox potential (starting with NADH or FADH2 and ending with O2)

• When electrons are passed from electron donor to acceptor, there is a release of free energy (potential energy has increased)

• Free energy stored in electrochemical gradient is dissipated through ATP synthase to drive ADPphosphorylation

Chemiosmotic Energy Conservation

• electrochemical gradient is dissipated through ATP synthase; free energy is used to drive ATP synthesis

• Transmembrane electrochemical proton gradient is a form of energy storage that is adaptable to many forms of energy utilization

• Protons go down concentration gradient through ATP synthase, driving reaction forward

• Conservation of energy

Processes in Mitochondria that use the Proton Motive Force

1. ATP synthase

2. Pyruvate transport by the MPC (pH gradient) (MPC: Mito Pyruvate Carrier)

3. Inorganic phosphate uptake by the PiC (pH gradient)

4. NA+ - H+ exchange (pH gradient)

5. K+ - H+ exchange (pH gradient)

6. Ca2+ uptake by the Ca2+ uniporter (membrane potential)

7. Protein import (membrane potential)

8. Transporters and ion channels utilize secondary active transport → use proton motive force

9. Each is a form of energy conservation

10. All the above processes occur across the IMM

Key Concepts of Energy Conservation by Chemiosmosis

1. Mitochondrial respiratory complexes are proton translocating redox enzyme systems

2. ATP synthase is a reversible proton pump

3. IMM have low proton conductance

4. Agents that increase proton conductance prevent energy conservation in mitochondria and uncouple substrate oxidation from phosphorylation (uncouplers)

5. IMM must contain transport proteins to permit the movement of adenine nucleotides, phosphate, and substrates and products of ox-red reactions in and out of mitochondria and to maintain osmotic stability (this costs some of the proton motive force)

Energy is Synthesized on Demand

• take in food/fuel

• Can store it or oxidize some of it

• Ox couples to phos of ADP → ATP

• Controlled by metabolic demand (ion pumps, heat, work)

Respiratory Control

• Electron transport driven by free energy available in electron carriers

• Electron transport is restricted by chemiosmotic gradient: electron transport can only proceed when the gradient is dissipated (if hole in the bucket is plugged, eventually water can no longer be poured in)

• In healthy mitochondria, electron transport keeps up with utilization of the energy stored in the gradient ie. with ATP hydrolysis leading to ADP transport into the mito matrix (more ADP = large hole size → faster water (electron transport) refilling)

• The electron transport chain applies constant pressure to maintain an electrochemical gradient → adding ADP causes more electron flow

• **Adding more electron transport chain and ATP synthase proteins will not necessarily increase ATP synthesis

• need to have some release of gradient or oxidation will stop

Where do NADH and FADH2 come from?

• NADH → glucose oxidation (glycolysis and CAC) and fatty acid oxidation

• FADH2 → succinate dehydrogenase (CAC) and fatty acid oxidation

• Glucose and fatty acid oxidation

• Glycolysis and citric acid cycle

Proton Leak and Uncoupling of Oxidative Phosphorylation

• Uncoupling by lipid soluble weak acid, uncoupling protein (UCP), adenine nucleotide translocase promotes uncontrolled electron transport and hydrolysis of ATP

• ANT exchanges ADP and ATP across the IMM, gets ADP into mito and ATP out → can dissipate electrochemical gradient

• UCP also mediate leaks and dissipates proton gradient and generates heat

• Weak acids (protonophors) can also penetrate the membrane and mediate leaks by carrying protons across membranes

• More leaks → more electron flux → more pull on substrate/demand

Uncoupling: Increasing Rate of Proton Leak through IMM

• Some H+ permeability of IMM is essential for chemiosmotic energy conservation

• Any factor that increases H+ leak rate will “uncouple” oxidation from phosphorylation

• Uncoupling activates electron transport independent of availability of ADP and Pi (can promoter hydrolysis of ATP as a consequence of dissipating the proton electrochemical gradient)

• Uncoupling proteins (UCP1) → membrane transporters that enhance H+ leak rate through IMM, but depend on binding of long chain FAs for activation

• UCPs are regulated by binding of guanine or adenine nucleotides

• UCP1 → only really good conductor of protons, only expressed in brown adipose fat (contains more mitochondria than white fat)

• UCP1 turned on by hydrolysis of FA (from norepinephrine) → activates GPCR → increase in cAMP → activates PKA → lipolysis via HSL → TG broken down into FA → uncoupling → generates heat

• ADT allows ADP entry into the matrix

Benefits of Expressing Uncoupling Protein

• Ensuring continue electron low → unrestricted substrate oxidation: promoting substrate removal unrestricted by the demand for metabolic energy

• Oxidative stress defense suppression of excessive mitochondrial formation of ROS (occurs via glut of reducing power)

• Thermogenesis: converting oxidative energy into heat

Efficiency: Organization of OxPhos Complexes

• Oxidative Phosphorylaion occurs in the cristae space

• MICOS Complex creates narrowing of cristae junction creating barrier for diffusion (diffusion limitation) → protons and cytochrome c cannot leave

• Cytochrome C released when junction is disrupted → signal for apoptosis

Respiratory Supercomplexes

• ETC complexes in intact mitochondria exist as loose aggregates along the crista wall → super complexes or respirators

• Respiratory chain super complex formation depends on the presence of supplementary subunits such as COX7A2- long/short and requires cardiolipin (IMM phospholipid)

• Advantages relate to efficiency (through channeling of redox intermediates or association with other mitochondrial components) or regulation of activities of subsections of the respiratory chain

• E- handed off more quickly/efficiently

Disrupted Respiratory Chain Supercomplexes

• Disrupted when cristae junctions loosen

• May facilitate ROS formation and cytochrome c release due to electron leakage