Lecture 28 notes Chapter 21 proton motive force_BCH400 spring 2025 v1 copy
Page 1: Introduction to Peter Mitchell and Chemiosmotic Theory
Peter Mitchell
Life: 1920-1992
Nobel Prize: 1978 for the Chemiosmotic Theory
Significant Concept: A proton concentration gradient serves as an energy reservoir for ATP formation.
Key Concept
Proton Motive Force (PMF): The driving force for ATP synthesis.
Page 2: Understanding Proton-Motive Force (PMF)
Definition of PMF
PMF is used to drive ATP synthesis by creating an electrochemical gradient.
It is a combination of:
Chemical Gradient (DpH): Represents the pH difference across the membrane (0.5 – 0.75) contributing 85% to energy.
Electrical Potential (DY): Ranges from ~140 to 170 mV.
Chemiosmotic Hypothesis
ATP synthesis is powered by the movement of protons across the membrane, based on the chemiosmotic hypothesis.
Page 3: Contributors to PMF
Key Contributors to Proton-Motive Force
Greatest Contributor: Charge gradient generated by protons.
Other Contributors:
Flux of pyruvate into the matrix
Rate of ATP synthesis
Concentration gradient of protons
Current from electron flow within the inner mitochondrial membrane
Page 4: Explanation of PMF Contribution
Determining Contributors
Charge and Concentration Gradients: Both contribute to the free energy associated with PMF.
DG = -2.303 RT DpH
Charge gradient contributes approximately 85% of the free energy.
Free Energy Calculations:
∆G for 1 proton through FoF1 = -19.4 kJ/mol.
∆G for ATP synthesis from ADP + P = +32 kJ/mol.
ATP Synthesis Calls for Multiple Protons
Must move more than one proton for ATP synthesis
Page 5: Evidence for Chemiosmotic Hypothesis
Experiments and Findings
Chemiosmotic Hypothesis Evidence:
ADP + Pi -> ATP requiring proton flow.
Page 6: Experimental Confirmation of PMF
Experiment Details
Artificial Vesicles: Created with bacteriorhodopsin and ATP synthase.
Bacteriorhodopsin: A light-activated proton pump. Jan
Findings: The ATP synthesis occurred due to proton pumping into the vesicle when illuminated, verifying the separation of the respiratory chain and ATP synthase, linked by proton-motive force.
Page 7: Mitochondrial Functioning Without Light
PMF in Dark Conditions
Mitochondria can operate and produce PMF independent of light; it comes from the Electron Transport Chain.
Summary of Components Involved:
ADP + Pi -> ATP
H+
Matrix & Intermembrane space roles in ATP production.
Page 8: Structure of ATP Synthase
Composition of ATP Synthase
F0 Component: Embedded in the inner mitochondrial membrane containing the proton channel (with 8 to 15 c subunits).
F1 Component: Contains active sites for ATP synthesis (three active sites on β subunits).
Components Connection: γ stalk connects F1 and F0, facilitating ATP production.
Page 9: Binding Change Mechanism of ATP Synthesis
Conformations of β Subunits
The three forms:
O (Open): Nucleotides can bind/releases.
L (Loose): Traps nucleotides.
T (Tight): Synthesizes ATP from ADP + Pi.
γ Subunit Role
Rotation of γ subunit interconverts the β subunits during ATP synthesis.
Page 10: ATP Release Mechanism
Mechanism of ATP Release
T-form converts ADP + Pi into ATP but doesn’t release it.
Rotation of γ subunit (120° CCW) converts T-form to O-form, allowing ATP release and binding of new ADP + Pi.
Page 11: Distinction between ATP Formation Mechanisms
Mechanisms Overview
Binding Change Mechanism: Indicates mechanical changes during ATP formation different from substrate-level phosphorylation (direct transfer of high-energy phosphate to ADP).
Page 12: Visualization of γ Subunit Rotation
Experimental Setup
Cloned α3β3γ subunits visualization of γ rotation during ATP hydrolysis.
Movement: Attachment of actin filament to γ subunit shows rotation as power is derived from ATP hydrolysis.
Page 13: Proton Flow Through ATP Synthase
c Ring Proton Movement
Protons move through the F0 component of ATP synthase, causing rotation:
Protons enter intermembrane space half-channel, bind to c ring glutamate/aspartate.
Rotation allows release into the matrix half-channel.
Page 14: Proton Flux and c Ring Operation
Mechanism Overview
Mechanism of Rotation: The neutralization of negative charge allows c ring subunits to rotate and produce ATP.
Page 15: Efficiency of Vertebrate ATP Synthase
ATP Production Efficiency
Vertebrate c ring: Composed of 8 subunits.
Proton Requirement: One full rotation requires 8 protons, producing 3 ATP molecules.
Significance: The number of c ring subunits directly affects ATP synthesis efficiency.
Page 16: Entry of NADH Electrons
Mechanisms for NADH Entry
NADH Transport: Requires mechanisms (shuttles) to transfer electrons from cytoplasm into mitochondria.
ATP Transport: Mechanisms also exist for newly synthesized ATP to exit.
Page 17: Glycerol 3-Phosphate Shuttle
Shuttle Functionality
Transfers electrons from NADH to FAD to QH2.
Important in specific metabolic contexts like insect flight muscles.
Page 18: Malate-Aspartate Shuttle
Functionality and Characteristics
Malate can cross the mitochondrial membrane, while oxaloacetate cannot.
Transfers electrons from NADH to another NADH in the mitochondrial matrix.
Page 19: ATP-ADP Translocase Mechanism
Role in Metabolite Exchange
ATP-ADP translocase: Exchanging one ATP out for one ADP in.
Various carriers for other metabolites require energy (~25% of oxidative phosphorylation energy).
Page 20: ATP Yield from Glucose Oxidation
ATP Production Overview
Out of ~30-32 ATP from glucose combustion:
26-28 via oxidative phosphorylation.
4 from substrate-level phosphorylation.
Fermentation yields only 2 ATP.
Page 21: Accounting for ATP Yield
ATP Formation Breakdown
Overview of contributions:
Substrate-level phosphorylation yields net 2 ATP.
NADH electrons originate from glycolysis in the cytoplasm.
Page 22: Major ATP Consumers in Eukaryotic Cells
ATP Consumption Categories
Processes consuming ATP:
30% for protein synthesis
25-35% for P-type ATPase ion pumps (Na/K and Ca ATPases).
Energy requirement to maintain body temperature is significant; ~20% of electron transport energy is uncoupled from ATP synthesis.
Page 23: Pyruvate and Electron Transport Shutdown
Experiment Insights
Mitochondrial experiments reveal that the ATP synthase ceases functioning when ADP is depleted.
O2 Consumption as Measure: Indicates electron flow is absent when proton gradient maxes out without ADP.
Page 24: Uncouplers and Electron Transport
Uncoupling Mechanisms
Uncouplers allow electron transport without ATP production by moving H+ across the membrane.
Historical Context: DNP was banned as a weight-loss drug due to its effects.
Page 25: Effects of 2,4-Dinitrophenol as an Uncoupler
Mechanism Overview
Uncouples oxygen consumption from ATP formation, keeping electron transport active by reducing proton gradient.
Page 26: Mammalian Uncoupling Mechanisms
Importance of Uncoupling Protein (UCP)
Mammals (excluding pigs) can uncouple electron transport to regulate body temperature.
Essential for newborns and hibernating animals.
Page 27: ATP Synthesis Inhibitors
Electron Transport Inhibitors
Key Inhibitors:
Rotenone
Cyanide
Azide
MPTP affects complex IV.
Page 28: Role of Complex IV
Functionality of Cytochrome c Oxidase
Complex IV catalyzes the reduction of molecular oxygen to water.
Toxicity of CO due to higher affinity for binding than O2.
Page 29: Understanding Rotenone
Characteristics
Plant-derived rotenonoid (from South America, etc.) that blocks Complex I.
Less effective in mammals due to absorption issues.
Page 30: Parkinson's Disease and MPTP
Connection to Parkinson's Disease
MPTP is a neurotoxin blocking Complex I, linked to synthetic heroin contamination.
Discovered link highlighted by Dr. Langston regarding its effect.
MPTP preferentially destroys dopaminergic neurons, crucial in Parkinson's pathology.
Page 31: Importance of Complex 1
Nutrient Metabolism Dependency
Complex I is essential as FADH2 cannot simply substitute without TCA cycle function.
TCA cycle needs NAD+ regeneration done by Complex I to function properly.