Electron Transfer Chain (11/3 & 11/8)

What are the four complexes involved in the ETC? Other enzymes that are involved?

1. Complex 1 • NADH dehydrogenase • Deals with incoming NADH and oxidizing it 2. Complex 2 • Involves Succinate DH (exact same enzyme in the TCA cycle) • Oxidizes FADH2 3. Complex 3 • Cytochrome type oxidase 4. Complex 4 • Cytochrome type oxidase 5. Other enzymes • Acyl-CoA DH (enzymes that catalyzes the first oxidation in beta oxidation, generating FADH2) • Glycerol-3-P DH (when making TAGs and breaking down glycerol to bring it into the glycolytic pathway)

What are uncouplers?

Biological and non-biological mechanisms that uncouple the ETC and ATP synthase

What are nonbiological uncouplers?

Chemicals that can uncouple the ETC and the ATP synthase

Describe the inputs and outputs of the REDOX reactions that are occurring in each complex in the ETC.

Complex 1: • Input: NADH • Output: Reduced Coenzyme Q (CoQ) • Reaction: NADH is oxidized, and CoQ is reduced in the process of electron transfer. Complex 2: • Input: Succinate • Output: Reduced CoQ • Reaction: Succinate is oxidized to fumarate, and CoQ is reduced. Complex 3: • Input: Reduced CoQ • Output: Re-oxidized CoQ, Reduced Cytochrome C • Reaction: CoQ is re-oxidized, and Cytochrome C is reduced; Two Cytochrome C molecules are needed because CoQ is a 1 + 1 (2) electron carrier, while Cytochrome C is a one-electron carrier Complex 4: • Input: Reduced Cytochrome C, Protons, Oxygen • Output: Re-oxidized Cytochrome C, Water • Reaction: Cytochrome C is re-oxidized, and oxygen is reduced to water • In these reactions, electrons are transferred through the complexes, reducing the next carrier, and the one that donates electrons is reoxidized • This happens step by step, and the reactions are doubled up, with 1/2 oxygen representing O2 • Complex 1 and Complex 2 operate independently of each other, with one entry point for NADH and one entry point for FADH2

What is generated during the ETC as REDOX reactions continue to occur?

Proton Motive Force (PMF) • Complexes 1, 3, and 4 take protons from the matrix and pump them into the intermembrane space, facilitated by the double membrane structure of mitochondria in eukaryotes • This process builds a higher proton gradient in the intermembrane space, creating an energy-storing potential • The protons, unable to freely flow back into the matrix due to membrane impermeability (ions cannot go through the hydrophobic inner membrane space) generate energy as they seek equilibrium • This proton gradient is essential for ATP synthesis and is utilized by the ATP synthase enzyme • The back-flow of protons back into the matrix through ATP synthase drives the phosphorylation of ADP to generate ATP

What will cause energy to increase in a proton gradient?

If the gradient is not released, that energy will go up • The more concentration gradient you have, the harder it will be to keep pumping protons • ANALOGY: if you pump up your tire, the more air you pump in, the harder it will be to pump up the tire; when you release the valve, the air will get out and could be used for biochemical work (bike tire is mostly heat, the valve will get hot)

What is the direction of rotation in the ATP synthase (i.e. how does the ATP synthase function in terms of rotation)?

In the F0 domain of ATP synthase, c-subunits form a rotor with long alpha helices, and one c-subunit is connected to a static alpha domain • An arginine is coming from an alpha domain which is bonded to a lysine sitting in one of the C-subunits of the rotor 1. Entry of a proton breaks the bond between lysine (c-subunit) and arginine (alpha domain) • Broken lysine-arginine bond causes lysine to flip away, releasing arginine 2. Released arginine binds to the lysine of the neighboring c-subunit 3. The lysine that accepted the proton gives it away on the matrix side of the membrane 4. This process repeats for each proton, leading to continuous rotation of the ATP synthase rotor • Note: Protons have a higher affinity for lysine and arginine than for the bond between these amino acids

Where does the ETC take place? Why is this advantageous?

Inner membrane of mitochondria • A vast majority of the catabolic pathways are in the mitochondria in the matrix (ETC has direct access to their reduced electron carriers); exception is NADH generated from the glycolysis which takes place in the cytosol • A membrane is needed to build a proton gradient

How do we get cytosolic NADH from glycolysis into the mitochondria to use it in the ETC?

Malate OAA Shuttle (MOA) • Works oppositely to when we transfer electrons from NADH in the mitochondria by reducing OAA to Malate, then transporting Malate to the cytosol to be re-oxidized in to OAA and transferring the electrons to cytosolic NAD+ to produce NADH • NADH from glycolysis is oxidized to NAD+ in order to reduce OAA to Malate • Malate is then transported into the mitochondria, where it is oxidized back to OAA, generating NADH in the mitochondria • This process enables the transfer of electrons from cytosolic NADH to the mitochondrial electron transport chain, contributing to ATP production • The MOA shuttle is a versatile mechanism used in both directions based on cellular energy needs, supporting either glucose synthesis or ATP generation

Why are the complexes in the ETC situated closely together?

Multiple ETC complexes are closely situated along the inner membrane to prevent electron loss (due to spontaneous reaction with a positive charge) and facilitate efficient electron transfer

What are sources for reduced electron carriers that go into the ETC?

NADH and FADH2 • Reaction 6 of Glycolysis • PDH reaction • TCA Cycle • Beta oxidation • Amino acid catabolism

What is the uncoupler protein (UCP)? What does it produce?

• Biological uncoupler • A channel 1. UCP Functionality • Positioned near the Electron Transport Chain (ETC), UCP allows protons to flow back across the membrane, circumventing the ATP synthase 2. Purpose of Uncoupling • By permitting the spontaneous back-flow of protons without ATP synthesis, UCP disrupts the usual coupling of electron transport and ATP production 3. Outcome • The energy released through this uncoupling process is not harnessed for ATP synthesis • Instead, the dissipated energy manifests as heat, converting the potential ATP production into thermal energy 4. Heat Generation • The primary aim of this uncoupling mechanism is to trade ATP production for heat generation

What is the chemoismotic theory?

• Building up a concentration gradient in this case of protons and using the energy in that to make ATP

What is the function of copper in proteins?

• Can be coordinated in proteins; can act as one electrons carriers

What is Coenzyme Q (Q)?

• Coenzyme Q (CoQ) (i.e. ubiquinone) is a non-protein molecule resembling a lipid • Contains a hydrocarbon tail and a polar end group; lipid-like nature allows it to move within the membrane dynamically • CoQ serves as an excellent intermediate between ETC complexes due to its ability to accept and donate electrons • In its oxidized form, CoQ has a quinone structure, with ketone functions in the polar head group, which can be reduced to hydroxy groups • CoQ can accept electrons either in pairs (from NADH) or individually (from FADH2); flexibility makes CoQ a central point for electron transfer in the ETC, efficiently gathering electrons from both complex 1 and 2 and facilitating their transfer to complex 3 • Where the branching ends in the ETC

What happens in Complex 2 of the ETC?

• Complex 2 of the electron transport chain (ETC) receives electrons from FADH2, with the source being the enzyme succinate dehydrogenase (succinate DH) • Succinate DH is a TCA cycle enzyme that operates downstream, converting succinyl-CoA to succinate in the TCA cycle (specifically in the step from succinate to fumarate) • This enzyme bridges the TCA cycle and the ETC, as it plays a role in both processes • Enzymes Acyl-CoA DH and Glycerol-3-P also bring in FADH2 to be oxidized into FAD • The electrons received from FADH2 (Succinate DH, Acyl-CoA Dh, and Glycerol-3-P) are transported to CoQ

What happens in Complex 4 of the ETC?

• Complex 4 will take some protons from the matrix, take oxygen, electrons from cytochrome C, and reduce oxygen into water • End of the ETC

What are hemes and where are they located?

• Electron carriers that are very prevalent in nature • Porphyrin rings which contains an iron complex in the middle of it • Iron and heme responsible for making our blood red (replace iron with magnesium yields green) • Iron sitting in the cytochrome C protein and is responsible for accepting electrons and donating them to another electron carrier • Carries one electron at at time

What is the primary reaction that occurs in the ETC?

• The electron transport chain (ETC) is a sequence of enzyme complexes and enzymes that facilitate the transfer of electrons through redox reactions • In this process, one complex donates electrons, leading to its oxidation, while the recipient complex undergoes reduction

How does Complex 2 function?

• The enzyme is anchored in the mitochondrial membrane, with one part facing into the matrix • Enzyme functions both in the TCA cycle and the ETC • Succinate, generated in the TCA cycle, is oxidized to fumarate in Complex 2 • The electrons produced in this process are transferred through iron sulfur clusters within the enzyme until they reach ubiquinone (coenzyme Q), reducing it to ubiquinol • Complexes operate by coordinating enough electron carriers within the protein, allowing electrons to hop from carrier to carrier • Specific arrangement of these carriers is crucial for efficient electron transfer • Note: the ETC is not 100% efficient, and some electrons are lost in the process • Examples of inefficiency of the complexes is if an iron sulfur cluster (or another electron carrier) were to be missed (e.g. one iron sulfur cluster would never get re-oxidized) or the electron would react with any positively charged amino acid

How fast is the ATP synthase rotor?

6,000 RPM (revolutions per minute)

What are the inhibitors that can block certain complexes in the ETC? What are the consequences of those inhibitors?

1. Barbiturates • Blocks Complex 1 • Dramatically slows down the ETC due to the inability for complex 1 to oxidize incoming NADH • ETC can still function due to complex 2 accepting electrons from FADH2 2. Antimycin A • Blocks Complex 3 • The ETC would no longer work since the ETC is no longer branched from CoQ onward 3. Cyanide • Blocks Complex 4 • Stops the WHOLE ETC and ATP production • Complex 4 is relevant for bringing the electrons to the final acceptor that's oxygen • Leads to suffocation at a molecular level, because we can't bring the electrons to oxygen. • In this case, electron transport would stop, if electron transport stops, we do not make a proton gradient anymore, and if we don't have that, we don't make ATP anymore›››¢

Where is the UCP present?

1. Brown adipose • Newborns have abundant brown adipose tissue (BAT) with high mitochondrial content, aiding heat retention in cold environments • As individuals age, both BAT and Uncoupler Protein (UCP) activity decline, reducing the capacity to generate heat through this mechanism 2. Hibernating animals • Use UCP to generate heat during hibernations • End of hibernation leads to switching back to full ATP production 3. Shrunk Cabbages • Utilize Uncoupler Protein (UCP) to generate heat, melting snow during cold temperatures • This early heat production allows them to bloom ahead of other plants, providing an ecological advantage in early pollination by attracting flies

What does coupling of the ETC and ATP synthase result in (in regards to SRP)? Why must they be coupled?

1. Direction of Electron Flow: • Electrons flow from lower Standard Reduction Potential (SRP) to higher SRP, corresponding to a lower Gibbs Free Energy (ΔG) • Understanding the series of events in the Electron Transport Chain (ETC) involves observing the sequence of electron flow based on SRPs 2. Proton Motive Force (PMF): • The energy released during electron transfer across ETC complexes contributes to the formation of the Proton Motive Force (PMF) • PMF is crucial for establishing a proton gradient across the inner mitochondrial membrane 3. Role of PMF in ATP Generation: • The energy stored in the PMF gradient is harnessed to generate ATP • The process linking electron transfer and ATP production is known as Oxidative Phosphorylation 4. Oxidative Phosphorylation: • Oxidative Phosphorylation is coupled to redox reactions, utilizing the energy released from oxidizing reduced electron carriers • These electron carriers originate from glycolysis and other metabolic pathways 5. Reasons for Coupling • When you DON'T release the proton gradient, that's when the energy will get higher • For not making ATP, or release the proton gradient, then the energy will increase • The gradient has to be FINELY balanced for the energy needed to pump protons and releasing it again to keep it at a certain level

What are common ways that the ETC can be elucidated?

1. Enzyme biochemical characterizations 2. Inhibitors that are specific to individual complexes 3. Isolation of the mitochondria 4. SRP (Standard Reduction Potentials)

How does the isolation of the mitochondria contribute to the elucidation of the ETC?

1. Isolation of Mitochondria: • Mitochondria are isolated, typically from liver tissue, by mashing it up The isolated mitochondria are placed in a solution, and the oxygen is removed by bubbling in nitrogen 2. Depletion of Oxygen: • NADH is added to initiate the ETC, providing electrons As oxygen is depleted, all complexes in the ETC become reduced because there is no final electron acceptor 3. Starting the Experiment: • The experiment begins when oxygen is reintroduced into the solution 4. Graphical Analysis: • A graph is constructed with time on the x-axis and the percent oxidation of the complexes (designated as X, Y, and Z) on the y-axis • At time point 0, oxygen is introduced. 5. Interpreting the Graph: • Steeper Curves: The faster oxidation of a complex (e.g., X) indicates it is the first to give electrons to oxygen Flatter Curves: Slower oxidation (e.g., Y and Z) follows, representing subsequent steps in the oxidation reactions 6. Kinetics and Series of Oxidation Reactions: • The kinetics of oxidation reveal the order in which complexes contribute electrons to oxygen • The analogy is drawn to a traffic light scenario, where the first car (complex X) moves first, causing a sequential movement of other cars (complexes Y and Z) 7. Notes • This experiment with redox kinetics provides insights into the dynamics of the ETC, showcasing the relative rates at which different complexes are oxidized when oxygen is reintroduced

How do NADH and FADH2 differ?

1. Solubility • NADH is soluble, allowing it to move freely between enzymes • FADH2 remains enzyme-bound at the ETC 2. Electrons • NADH transfers two electrons simultaneously to the next acceptor, always in pairs • FADH2 has the flexibility to donate electrons individually; this characteristic enables FADH2 to interact with both one- and two-electron carriers in the ETC, providing dynamic flexibility in electron transport (1 + 1)

How do you calculate the SRP of an individual complex within the ETC (e.g. Complex 1)?

1. Specific Complex Analysis: • For Complex I (NADH to CoQ):SRP for the oxidation of NADH is 0.32.SRP for the reduction of CoQ is 0.06 • The summed SRP for this complex is 0.38 V 2. Energetic Contribution of Complex I: • Applying the Gibbs Free Energy equation yields an energy release of about -70 kJ/mol for Complex I This indicates that Complex I, in the context of NADH to CoQ, contributes to the overall energy release in the ETC 3. Comparison to Overall ETC: • The overall ETC releases about 220 kJ/mol. While Complex I accounts for -70 kJ/mol, it is part of the larger picture of energy release across the entire ETC 4. Relation Between SRP and Gibbs Free Energy: • The higher the SRP (ΔEº'), the more negative is the Gibbs Free Energy • A more negative Gibbs Free Energy indicates a higher reduction potential and more energy being released 5. Prediction of Proton Pumping: • The energy released during individual complex reactions is utilized to pump protons across the inner mitochondrial membrane • Higher reduction potentials suggest more effective proton pumping 6. Theoretical Addition of SRPs: • Theoretically, the SRP of each individual complex should add up the SRP of the oxidation of NADH and the reduction of O2 • This theoretical sum helps predict the cumulative effect of each complex in the electron transport process

Why is Complex 2 only complex that cannot contribute to the proton gradient?

1. Standard Reduction Potential (SRP) and Energy Release: • Complexes 1, 3, and 4 pump protons, contributing to the establishment of the Proton Motive Force (PMF) for ATP synthesis • Complex 2 releases less than 19 kJ/mol of energy during electron transport and does not pump enough protons • Energy release during electron transport must exceed 19 kJ/mol for effective proton pumping 2. Energetic Requirements for Proton Pumping: • The free energy released during electron transport, coupled with proton pumping, should remain below zero (negative) to sustain the Electron Transport Chain (ETC) • If the Gibbs Free Energy becomes equal to or higher than zero, the ETC stops

How are SRP and Gibbs Free Energy related?

Gibbs Free Energy Connection: • Standard Free Energy (ΔG°) is related to SRP through the equation: ΔG° = -nFΔE° • ΔG° is the Gibbs Free Energy change • n is the number of electrons transferred (2 in this case). • F is the Faraday constant (96.5 KJ/mol V) ΔE° is the standard cell potential (SRP) • A more positive SRP yields a more negative Gibbs Free Energy

What is the primary role of reduced electron carriers in cellular respiration?

Reduced electron carriers, such as NADH and FADH2, capture the energy from the breakdown of energy-rich metabolites (e.g., carbohydrates, lipids) and transport this energy to the Electron Transport Chain (ETC) for ATP synthesis

How can SRP values be used to predict electron transfer in the ETC?

SRP is based on the measurement of voltage in a redox system, specifically involving electron carriers • It is used to predict the order in which electrons will flow through the ETC 1. Experimental Setup: • Two beakers are used • Left (Reference Beaker): Contains hydrogen gas equilibrated with protons, set as the reference at zero • Right (Sample Beaker): Contains a mixture of one molar concentration of the oxidized and reduced forms of an electron carrier (e.g., NAD+/NADH) • Connection with Salt Bridge: A salt bridge (usually potassium chloride solution) connects the two beakers, enabling electron flow, measurable as voltage 2. Measurement and Interpretation: Electron flow direction (from reference to sample or vice versa) determines the electron carrier's tendency to donate or accept electrons • If electrons flow from the reference to the sample: The sample is an oxidizing agent (accepting electrons); the SRP is positive • If electrons flow from the sample to the reference:The sample is a reducing agent (donating electrons); the SRP is negative 3. Implications of SRP: The strength of reduction or oxidation varies among electron carriers; electrons flow from a carrier with a lower SRP to one with a higher SRP 4. Prediction of Electron Flow: By measuring voltage and assigning SRP values under standard conditions, the order in which electrons flow from one carrier to another can be predicted • The SRP provides insights into the relative tendencies of electron carriers to undergo reduction or oxidation • In summary, the SRP is a tool that allows for the quantitative prediction of the direction of electron flow in redox reactions

What is the structure of the ATP synthase?

The ATP synthase consists of distinct domains: 1. F-Zero (F0) Domain • Located at the bottom of the enzyme in the intermembrane space • Composed of subdomain A and multiple C-subunits forming a rotor • Contains a proton-conducting tunnel allowing protons to move from the intermembrane space into the matrix • Referred to as the rotor, as it rotates during the ATP synthesis process 2. Shaft (Gamma Domain) • Connects the F0 domain (rotor) to the F1 domain • Facilitates the transmission of rotational energy from F0 to F1 3. F1 Domain • Located at the head of the ATP synthase • Consists of a mixture of three alpha and three beta domains • ATP synthesis takes place in this region 4. Additional Domains • Other domains, such as b2, contribute to holding the ATP synthase in place • 20 nm in length • In essence, the F0 domain, embedded in the membrane, allows protons to flow back into the matrix, driving the rotation of the rotor and the transmission of energy to the F1 domain where ATP is synthesized • The overall structure of ATP synthase facilitates its role in the generation of ATP through oxidative phosphorylation

What is the SRP of the entire ETC?

The electron transport chain (ETC) involves a comprehensive series of redox reactions, and understanding these reactions in terms of half-cells and Standard Reduction Potential (SRP) is essential 1. Overall Reaction: • NADH + H+ is oxidized, and oxygen is reduced to form water • This full reaction can be broken down into two half-cells 2. Half-Cell Reactions: • Oxidation of NADH: Removal of two electrons and two protons • Reduction of Oxygen to Water: Addition of two electrons and two protons 3. SRP Measurement: • SRP values for each half-cell are determined experimentally • For the oxidation of NADH, the SRP is +0.32 (flipped from the reduction of NAD+) • For the reduction of oxygen to water, the SRP is 0.816. 4. Overall SRP Calculation: • SRP values for the two half-cell reactions are added • Overall SRP for the total reaction is 1.136 V, indicating electron flow between the two half-cells 5. Energetics of Electron Flow: • Calculating ΔG° for the overall reaction reveals -220 kJ/mol • This negative value indicates energy released during the electron flow from NADH to oxygen • This released energy is crucial for pumping protons, driving the proton gradient across the inner mitochondrial membrane

What is the function of the ATP synthase?

The protons that were pumped into the inter membrane space will flow back through the ATP synthase into the matrix • Protons going into the ATP synthase will drive rotation of part of the enzyme and that drives ATP production

What if there is an overdose of 2,4-DNP (i.e. overdose of biological and nonbiological uncouplers)?

There will be too little proton gradient to drive ATP production • The ETC cannot run and we cannot produce ATP

Why is Glycolysis and the TCA indirectly dependent on oxygen?

While glycolysis and the TCA cycle do not directly involve oxygen, they indirectly depend on it because they rely on the electron transport chain (ETC) • ETC requires oxygen to function • The ETC stops if oxygen is absent because oxygen serves as the terminal electron acceptor • Without oxygen, the ETC cannot work, and ATP production ceases

What is the glycerol-3-phosphate shuttle?

• Happens in specific organs and tissues (e.g. muscle and brain) 1. The electrons from NADH are then transferred to DHAP, creating a hydroxy group ultimately generating the metabolite glycerol-3-phosphate 2. Glycerol-3-phosphate can cross the outer mitochondrial membrane, and its electrons are transferred to FAD, generating FADH2 3. The electrons from FADH2 are eventually passed on to Coenzyme Q in the electron transport chain • Shuttle operates in a cyclic manner, allowing the continuous entry of NADH into the process. • Disadvantage: a slight reduction in ATP production compared to the Malate Oxaloacetate Shuttle, as FADH2 produces 1.5 ATP, while NADH contributes 2.5 ATP when entering the electron transport chain (1 less ATP)

Why was 2,4-DNP used to lose weight?

• If you uncouple the proton gradient, but we still want to make enough ATP • If we want to make enough ATP, we have to generate MORE protons • To generate more protons, we have to oxidize more NADH and FADH2, we have to break down more of our FATS and more of our GLYCOGEN

How does the rotation of the ATP synthase function?

• In an experiment, ATP synthase was heterologously expressed and tagged for visualization purposes. • Two types of tags were used: 1. Histidine Tags • Attached to the alpha and beta domains of ATP synthase • Histidine has a strong binding affinity to nickel complexes • Used to bind and place the enzyme on a surface 2. Biotin and Avidin Tags • Biotin was attached to ATP synthase • Avidin, a protein with strong binding to lipid-like structures, was used to visualize the tagged ATP synthase in a microscope • The main goal of the experiment was to demonstrate that ATP synthase functions as a rotor 3. Presence of Rotation • By unhooking the F0 domain and introducing ADP in the absence of a proton gradient, the ATP synthase started synthesizing ATP • This synthesis was accompanied by the observed rotation of the actin filament filler, providing evidence that the ATP synthase acts as a rotor, and its rotation is connected to proton flow

What are iron sulfur clusters?

• In the electron transport chain (ETC), electron carriers are often bound within complexes • Iron-sulfur clusters (e.g. Fe2S2 and Fe4S4) accept 1 electron at a time • These clusters consist of iron atoms complexed with cysteine residues in a protein and can have different configurations • Despite their structural variations, they all function as one-electron carriers. The significance of CoQ being a 1 + 1 electron carrier lies in its ability to interact with iron-sulfur clusters, allowing it to donate one electron at a time to these clusters for efficient electron transfer in the ETC • Multiple iron-sulfur clusters are present in the ETC.

What is oxidative phosphorylation?

• Involves using the energy from the oxidations in the electron transport chain (ETC) to create a proton gradient (proton motive force) • The impermeability of the membrane for protons forces them to flow back into the matrix through ATP synthase, leading to ATP production • The ETC and ATP synthase are tightly coupled; the ETC builds the proton gradient, and ATP synthase uses it to generate ATP • If either process is halted, the other cannot function effectively, emphasizing their interdependence

What are the electron carriers involved in the ETC?

• NADH • FADH2 • CoQ (ubiquinone, Q) • Iron Sulfur clusters (e.g. Fe2S2) • Hemes (sitting inside cytochromes) • Copper

What happens in Complex 1 of the ETC?

• NADH enters the electron transport chain (ETC) at complex 1 • Electron carriers in complex 1 are reduced while NADH is oxidized to NAD+ • NADH utilized in the ETC is derived from various pathways, including glycolysis, the PDH (pyruvate dehydrogenase) complex, the TCA cycle, beta-oxidation, and amino acid catabolism • Regardless of their origin, all NADH molecules enter the ETC at complex 1 • Subsequently, the electrons from complex 1 are transferred to the CoQ (Q) electron carrier

What happens in Complex 3 of the ETC?

• No more additional entry points for electrons from reduced electron carriers (NADH, FADH2) • Complex 3 will transfer electrons from CoQ to cytochrome C • Cytochrome C will transfer electrons over to complex 4

What is 2,4-Dinitrophenol (DNP)?

• Nonbiological uncoupler • A pesticide • When ingested, it is in its deprotonated form in the intermembrane space • Able to take a proton and become protonated, and can go across the membrane through free diffusion into the matrix • Accumulation of protonated 2,4-DNP ultimately disrupts the proton gradient • Aspirin functions similarly (not as severe)

How does the rotation of the ATP synthase link to the synthesis of ATP?

• Rotation of gamma domain (i.e. shaft) changes the conformation of the three beta domains • ß-domains are where ATP synthesis happen; can undergo three different conformations depending if it is in contact with the shaft or not • Alpha-domains act as structural support to hold the ß-domains in place; essential as they allow ß-domains to change conformation when hit by the shaft instead of simply moving • The rotation of the shaft drives a conformational change of these beta domains; that conformation change of the shape in the domain is what is driving ATP production • The shaft is not straight and symmetrical, it is sort of off shaped; means as the shaft is rotating, it's hitting all these beta domains in different ways • Depending on the position of the shaft it may hit ONE beta domain while not hitting another, causing one beta domain to change shape while another one stays the same • The three conformations that the three ß-domains can undergo are (in order): 1. Empty or Open - inactive conformation that has little affinity to bind to its substrate (ADP) 2. Ligand or Loose (L) - where the beta subunit accepts ADP and phosphate 3. Tight (T) - where ATP synthesis is actually happening • Each ß-domain undergoes each of the three conformational changes per round • Note 1. One rotation provides 3 ATP (conserved) 2. C-subunits not conserved; a larger rotor equals more protons needed per revolution 3. More protons needed means more NADH and more FADH2 is needed to drive the gradient; more resource to drive the ATP synthase

What is the function of Cytochrome C?

• Sits between complexes 3 and 4 • Similar to CoQ, but it is instead sitting within a protein • Can accept electrons from complex 3 by attaching to the complex and then move a little bit in the membrane to make contact with complex 4 • Once cytochrome C reaches complex 4, it donates the electrons • After cytochrome C donates electrons to complex 4, it flops back to complex 3

How do we transport ATP out of the cytosol, and ADP + inorganic phosphate into the cytosol?

• Transporting ATP in the cytosol allows it to be used in pathways such as glycolysis • The proton gradient provides energy in the form of an electrical differential/charge differential which is driven by oxidation • Transportation of ATP, ADP, and Pi is as follows: 1. To export ATP from the mitochondrial matrix and import ADP for regeneration, an antiporter called the Adenine Nucleotide Translocase (ANT) is employed 2. The proton gradient, characterized by both concentration and electrical charge differentials between the intermembrane space and the matrix, is utilized 3. ATP, being more negatively charged due to an additional phosphoryl group, exhibits a higher affinity for the intermembrane space with a more positive charge 4. ANT leverages this charge differential within the proton gradient to export ATP to the intermembrane space while concurrently importing ADP into the matrix 5. The exchange of charges across the proton gradient is a crucial mechanism for the continuous movement of ATP and ADP 6. Additionally, a symporter known as the Phosphate Translocase facilitates the influx of inorganic phosphate and utilizes a small portion of the proton gradient to channel protons through the membrane 7. The backflow of positively charged protons through this symporter aids in transporting a phosphate ion into the matrix Usage of the proton gradient can be used to achieve: • Making ATP • Bringing in ADP substrate inside while releasing ATP outside the matrix • Bringing inorganic phosphate