Topic 6 - Bioenergetics Flashcards
Key roles of mitochondria?
- A central player in bioenergetics
- Produce 90% of cellular energy
- Regulate cellular redox states
- Calcium homeostasis
- Produce reactive oxygen species
- Synthesis and degrade high energy biochemical intermediates
- regulate cell death –> programmed cell death
What are the requirements for the existence of life?
- Information –> Nucleotides/DNA
- Energy –> ATP
Both of these requirements are needed because life is improbable as living systems are highly ordered –> doesn’t follow 2nd law of thermodynamics.
Information specifies what form the order should take and energy drives the reactions and processes for it to occur.
How do we define energy?
In biochemistry –> The ability to cause specific change.
What are the different categories of change?
- Synthetic –> changes in bonds –> i.e. to make macromolecules.
- Mechanical –> muscle contraction/flagellum/ribosome movement
- Concentration –> change in concentration across a membrane.
- Electrical –> movement of ions across membranes (neurons)
- Heat –> heat production –> maintain temperature.
What are the different sources of energy?
- Solar radiation –> plants use to photosynthesize –> produce nutrients –> consumed by humans.
- Electrical discharge
- Chemical energy
What are the two ways chemical energy can be released?
- Fermentation –> glycolysis
- Oxidation –> respiration
Definition of thermodynamics?
The laws governing energy transactions that accompany most processes and reactions.
Definition of bioenergetics?
Applied thermodynamics –> Application of thermodynamic principles to reactions and processes in biological worlds.
Difference between classical and non-classical thermodynamics?
- Classical –> Equilibrium reactions in closed systems.
- Non-classical –> Does not occur at equilibrium, takes into account time and occurs in open systems.
Note –> Living systems do not operate at equilibrium –> hence we use non-classical thermodynamics.
What do living systems do when they consume nutrients? (In terms of enthalpy and entropy)
Living systems consume high enthalpy/low entropy nutrients and convert them to low enthalpy/high entropy products.
- Free energy from this is used to do work and thus allows organisms to create a high degree of organisation.
Why is it important for an organism to stay in a non-equilibrium state?
- In order to perform useful work
- Equilibrium processes cannot be directed –> all regulatory functions require a non-equilibrium state.
- A non-equilibrium state is Inherently unstable –> gives rise to biochemical reactions –> allow us to degrade.
Are regeneration and degradation constantly occurring?
Happens all the time –> requires a constant influx of energy.
What is one thing that always occurs in non-equilibrium reactions?
In non-equilibrium reactions, something must always flow.
Flow –> change in spatial distribution of something (matter/heat/electrical charge/etc.)
Note –> All these flows are conjugate to their thermodynamic force.
Examples
- Flow of matter in diffusion –> driven by conc. gradient
- Transport of heat –> temperature gradient
- Migration of electrical charge –> current –> due to voltage gradient
- Chemical reaction –> difference in chemical potential.
Can a thermodynamic force also promote a non-conjugate flow?
Yes, a thermodynamic force may also promote a non-conjugate flow.
For example, a concentration gradient of matter can give rise to a chemical reaction (or heat or electrical current)
I.e. ATP synthase uses an electrochemical gradient to create chemical bonds.
What is energy transduction?
Energy transduction –> when a thermodynamic force stimulates a non-conjugate flow.
What is metabolism?
Metabolism is the study of energy flow in biological systems –> overall process by which living systems acquire and utilize free energy.
Definition of exergonic reaction?
An exergonic reaction refers to a reaction where energy is released –> ΔG is negative.
Definition of endergonic reaction?
A chemical reaction in which energy is absorbed.
ΔG > 0
What are the names of the different molecules after ATP hydrolysis?
ATP –> ADP –> AMP –> Adenosine.
Why does the hydrolysis of ATP release so much energy?
Phosphoanhydride bond is a high energy bond which releases a lot of energy.
What is the ΔG for the hydrolysis of one phosphate and for two phosphates?
H2O + ATP —-> ADP + Pi (ΔG = -30.5 KJ mol-1)
ATP + H2O —-> AMP + PPi (ΔG = -45.6 KJ mol-1)
What factors impact the ΔG of ATP hydrolysis?
Depends on pH, ionic strength and [Mg2+]
Is it possible to have different nucleotide triphosphates?
Yes, some biosynthetic reactions are driven by the hydrolysis of other nucleoside triphosphates –> I.e. GTP.
What are phosphoryl transfer reactions?
Refers to the transfer of a phosphate from one compound to another.
R1-O-PO32- + R2-OH —> R-OH + R2-O-PO32-
A very important reaction for biosynthesis of proteins, nucleic acids and carbohydrates.
What is phosphoryl transfer potential?
Phosphoryl transfer potential –> examines the ease at which a phosphate group can be lost from a molecule. It is a relative measure (Phosphoryl transferred from a compound to water).
Greater phosphoryl transfer potential –> more energetically favourable for the hydrolysis of the phosphate group.
Why does ATP have a high phosphoryl transfer potential?
High phosphoryl transfer potential is due to the structural differences between ATP and it hydrolysis products.
- Resonance stabilisation –> Products (inorganic Pi has greater resonance stabilisation)
- Electrostatic repulsion –> negative charges close together in ATP molecule.
- Entropy increase –> entropy of products is greater
- Stabilisation due to hydration –> H2O stabilizes products –> makes reverse reaction less favourable.
What are the free energy sources in biological systems?
- Phosphate anhydride
- Enol Phosphate
- Some thioesters
- Some compounds containing N-P bonds –> phosphoguanidines.
How do you show a high energy bond in a molecule?
~ –> indicates high energy bond.
How to classify low energy and high energy phosphate compounds?
ATP ≈ -30 KJ mol-1
More than -30 KJ mol-1 (more negative) –> high energy phosphate compound
Less than -30 KJ mol-1 (more positive) –> low energy phosphate compound
How can the synthesis of ATP occur from ADP if ΔG is positive?
Use high energy compounds and couple the reactions in order to synthesize ATP.
What is substrate level phosphorylation?
The transfer of the phosphoryl group from a compound with a large ΔG value to ADP yielding ATP.
Why is ATP important?
- ATP is intermediate in energy –> serves as an energy conduit between super high energy and low energy phospho-compounds.
- The highly exergonic phosphoryl transfer reactions of nutrient degradation are coupled to the synthesis of ATP from ADP + Pi
Processes that require the consumption of ATP?
- Early stages of carbohydrate breakdown to produce low energy phosphate compounds.
- Interconversion of nucleoside triphosphates.
- Muscle contraction and active transport against the concentration gradient.
- Phosphoanhydride cleavage followed by pyrophosphate cleavage provides extra energy for fatty acyl-CoA synthesis and nucleic acid biosynthesis.
What are the two ways in which ATP can be formed?
- Substrate level phosphorylation
- Oxidative phosphorylation
What does adenylate kinase do?
Phosphotransferase enzyme that catalyzes the interconversion of adenine nucleotides (ATP, ADP, and AMP).
2ADP <—-> ATP + AMP
What is the rate of ATP turnover?
Note –> Atp is a free energy transmitter, not a reservoir.
- ATP pool supplies energy for about 1 minute
- At rest –> the average human turns over ATP at a rate of approximately 1.5kg/hour
- The brain only has a few seconds supply of ATP.
What molecule acts as a reservoir of ATP?
Phosphocreatine acts as a reservoir for ATP formation (muscle and nerve cells).
ATP + creatine <—–> Phosphocreatine + ADP
Why is the adenylate kinase reaction important in cells?
2ADP <—–> ATP + AMP
When we exercise large quantities of ATP are used up which results in high ADP concentration and low ATP concentrations.
Normally, ATP concentrations in cells are very low but when the reaction above is activated during exercise –> AMP concentrations increase –> this activates al energy producing pathways (glycolysis) –> Hence, AMP acts as a signal of low energy which stimulates the oxidation of nutrients.
What are NAD+ , FAD and CoA?
They are all ATP derivatives.
Note –> All nucleotide triphosphates are energetically equivalent but ATP is the primary cellular energy carrier.
How many electrons are needed to fully oxidise O2?
4 electrons
What is the most important physical property of the inner mitochondrial membrane?
It is impermeable –> nothing can simply diffuse through.
What are the 3 stages of energy extraction from food?
- Large molecules are broken down, absorbed and distributed –> no useful energy produced.
- Break down molecules into simple units –> acetyl unit of acetyl CoA.
- ATP is produced by complete oxidation of the acetyl unit.
Why is phosphate so important in biological systems?
- Phosphate esters are thermodynamically unstable but kinetically stable
- Stability due to negative charges that make them resistant to hydrolysis without the action of enzymes –> enzymes can then manipulate energy release –> results in regulatory molecules (Kinases and phosphatases)
- No other element has the same properties and it is also abundant.
Equations for the reduction of NAD+ ?
Equations for the reduction of FAD?
What is the role of the electron carriers (NADH and FADH2)?
The electron carriers capture electrons and thus store the cell’s reducing power.
What are the steps in glycolysis?
Where does glycolysis take place?
Cytoplasm
Can glycolysis take place in both aerobic and anaerobic conditions?
Yes, it can take place in both environments.
What are the steps in the Krebbs cycle?
Before cycle –> Pyruvate dehydrogenase transfer pyruvate to CoA to form acetyl-CoA.
- Acetyl-CoA donates 2 carbons to the cycle –> from pyruvate added to oxaloacetate –> enzyme citrate synthase.
- a) Dehydration reaction (water released) –> Enzyme Aconitase (contains an Fe and S atom)
- b) Hydration (Water taken in) –> enzyme aconitase
- Oxidative decarboxylation (1st) (NADH+H+ released) –> enzyme isocitrate dehydrogenase –> requires NAD+ as a coenzyme.
- Oxidative decarboxylation (2nd) (NADH+H+ released) –> releases CO2 –> enzyme Isocitrate dehydrogenase –> Utilizes co-enzyme A.
- Substrate level phosphorylation (GDP to GTP which is latter enzymes convert to ATP later) –> Enzyme succincyl - coenzyme synthase –> Note enzyme CoA is regenerated.
- Dehydrogenation –> Succinate dehydrogenase (dependent on FAD –> hence FADH2 is created)
- Hydration (Water taken in) –> Fumerase
- Dehydrogenation –> Malate dehydrogenase –> reuqires to presence of NAD+ so NADH +H+ is released.
Note –> Steps and picture don’t always match.
What are the main products of the Krebbs cycle?
Main products –> per pyruvate
3 x NADH
1 X FADH2
They generate reducing power.
How is the citric acid-regulated?
- Operates when ATP is needed
- High levels of ATP and/or NADH inhibit the citrate synthetase (first step of cycle).
- Conversely –> high levels of ADP and or NAD+ activate isocitrate dehydrogenase.
- Low levels of ATP or high levels of acetyl CoA speed up the cycle to give energy.
How does calcium impact the citric acid cycle?
Citric acid cycle is controlled by levels of Ca2+ concentration in the matrix.
For example…
High concentrations in the cytosol caused by an increase in muscle activity increase Ca2+ levels in the matrix which activates enzymes for the citric acid cycle.
Why are redox reactions important in biological systems? What factor influences an organisms ability to carry out redox reactions?
Using redox reaction we can use the transfer of electrons between species to do useful work.
The ability of an organism to carry out redox reactions depends on the redox state of the environment or its reduction potential.
What is reduction potential?
The reduction potential is a measure of the tendency of a chemical species to acquire electrons and thus be reduced.
It is measured in Volts, millivolts or E (1mv = 1 E)
The more positive the potential is –> the greater the species affinity for electrons is and thus has a greater tendency to be reduced.
In made direction do electrons flow (reduction potential)?
Electrons flow from the low to high reduction potential.
What is the standard reduction potential?
Standard reduction potential is measured under standard conditions and is defined relative to a standard hydrogen electrode –> which is arbitrarily given a potential of 0 volts.
What does the electron transport chain look like?
Electron transport chain is located in the inner mitochondrial membrane.
- Complex 1
In the membrane, you find Quinine –> highly hydrophobic molecule that is dissolved and diffuses freely in the inner membrane space.
- Complex 2
- Complex 3
Note between Complex 3 and 4 there is a hydrophilic cytochrome c molecule located on the outside of the membrane.
- Complex 4
What occurs in complex 1 (NADH dehydrogenase)?
1. NADH donates two electrons to complex 1. They are accepted FMN (FMN + 2H+ + 2e- —> FMNH2)
2. FMN donates one electron to FeS (it can only accept one electron). –> Results in the formation of quinone FMNH. (Note there are multiple FeS entres –> mammalian complex 1 has 7)
3. The FeS centres donate the electrons to quinine –> this is possible as there are quinine pools located in the non-polar region of the membrane.
4. Quinine gets reduced by complex 1 and acts as an electron carrier to complex 3 (Note –> it hangs around till it is fully reduced.)
What is FMN?
What is FMN?
- A prosthetic group of flavoproteins which is like FAD without the adenine nucleotide.
- FMN can accept two electrons from NADH
- But FMN can only give away one electron at a time –> results in the formation of a radical intermediate called a semiquinone.
What is FeS?
What is FES?
Iron-sulfur centres which are prosthetic groups to proteins (each has 1-4 iron atoms). They can only transfer one electron but there are multiple centres as mentioned.
Note –> cytochromes have a haem prosthetic group which absorbs light –> absorption of light can determine whether oxi- or red- occurs.
What is a quinine?
Quinine (also known as CoQ, Q, ubiqionone and Q10)
- Reduced to quinol in a 2 electron reduction reaction
Q + 2e- + 2H+ —> QH2
- It is an extremely hydrophobic molecule so it dissolves within the fatty acid part of the membrane —-> thus it can diffuse freely along the plane of the membrane.
What happens in complex III?
Complex III –> CoQ, cytochrome c reductase
- Reduced Q donates 2 electrons into complex III
- One electron goes to FeS and the other goes to a low-affinity cytochrome b.
- a) FeS donates its electron to cyt C1
b) The electron is transferred from the low to high-affinity cytochrome b and subsequently returned to Q. - cyt C1 donates its electron to cytochrome C.
- Cytochrome C acts a mobile electron carrier thatis reduced by Complex III and oxidised by complex IV.
Explain the structure of complex I.
Complex 1
- Complex 1 is L shaped with a large hydrophobic portion and a large hydrophilic portion
- The Q chamber which is long, narrow and enclosed binds the two regions
- Q chamber is linked to one channel through a funnel of charged residues which continue along the entire membrane domain.
- The polar residues become surrounded by water to form a continuous hydrophilic axis spanning the entire domain
- The hydrophobic region has many vertical half channels made of vertical helices
- One set is exposed to the matrix, the other to the inner membrane space
- Horizontal helices connect to vertical helices on matrix side and β hairpin (βH) helices link the vertical helices on the inner membrane space.
Explain the structure of ATP synthase.
ATP synthase
- ADP + Pi –> ATP –> Synthesis of a phospho-anhydride bond
- F1 (segment) is in the matrix and F0 (segment) is in the membrane.
- There is a stalk region which interconnects the subunits.
-
F1 catalyses ATP hydrolysis
- F1 (α3β3γδε)
- Made up of homologous α &β (both have different conformations) subunits which alternate in a ring around the γ centre/stalk
- Three nucleotide binding sites are present at αβ interfaces, mainly involving β subunit residues
- γ creates asymmetry –> different interactions with α and β subunits
- β contains the catalytic site for ATP synthesis.
- Mg2+ will bind to the nucleotides in all the subunits
- ATP synthase, without the presence of a proton gradient, will catalyse the reverse reaction and hydrolyse ATP to ADP
5. F0 (a1b2c9-10)
- The membrane containing F0 is leaky to protons however inhibitors can interact with F0 to block the leaky properties
- Number of c depends on the species
- Also contains accessory peptides e.g. d, F6 and OSCP
Step by step process of how thermogenin is activated?
- Noradrenaline (controls fatty acid concentration in brown adipose) binds to a receptor.
- Adenylate cyclase increase cAMP
- cAMP activates cAMP dependant protein kinase
- The kinase phosphorylates triacylglycerol lipase –> activating it
- Lipase hydrolyses the triacylglycerol into fatty acids
- Activates and opens the Thermogenin proton channel.
- Protons do not enter ATP synthase so does not create the electrochemical gradient
- Heat is produced.
How is ATP/ADP transported in and out of the mitochondria?
ATP and ADP can not pass inner-membrane –> highly charged.
Transported via a ATP/ADP translocase –> Can be used for either ATP or ADP –> when they bind there is a conformational change which moves them across the membrane.
- Acts as an antiport –> exchange of ADP and ATP (ADP can only enter if ATP exists)
- Note –> inner membrane has a positive membrane potential –> ATP has one more negative charge than ADP –> So ATP out and ADP in is favoured.
- The exchange is driven and uses up the membrane potential –> hugely expensive process uses up a 1/4 of the proton-motive force generated by the respiratory chain.
How can we transfer electrons from the cytosol to the mitochondria?
Malate-aspartate shuttle –> Common in heart and liver cells.
- The function is to transfer electrons of cytosolic NADH to mitochondrial NAD + to form NADH.
- Mitochondrial NAD + is reduced by cytosolic NAD+ through the reduction and regeneration of oxaloacetate.
- Each NADH yields just over 2 ATP
- The carrier proteins have no NADH interactions, only with electrons moving into the matrix.
Process
Cytosol –> Oxaloacetate reduced by malate NADH to form Malate (transported via protein into Mito)
Mitochondria –> Malate oxidised to form NADH and oxaloacetate.
- The rest of the reactions in the cycle are present to regenerate oxaloacetate
What two stages can the glycolytic pathway be divided into?
Stage 1 is the trapping and preparation phase. No ATP is generated in this stage.
In stage 1, glucose is converted into fructose 1,6-bisphosphate in three steps: phosphorylation, an isomerization, and a second phosphorylation reaction –> function: trap the glucose in the cell and form a compound that can be readily cleaved into phosphorylated three-carbon units.
Stage 2: ATP is harvested when the three-carbon fragments are oxidized to pyruvate.
The function of hexokinase in glycolysis? (Step 1)
Glucose enters the cell via glucose transporters.
Hexokinase phosphorylates glucose to form glucose-6-phosphate.
Reason for this:
Glucose 6-phosphate cannot pass through the membrane because of the negative charges on the phosphoryl groups, and it is not a substrate for glucose transporters.
Addition of the phosphoryl group facilitates the eventual metabolism of glucose into three-carbon molecules with high-phosphoryl-transfer-potential.
Explain the isomerisation of glucose-6-phosphate to fructose-6-phosphate in glycolysis? (step 2)
Isomerization of glucose 6-phosphate to fructose 6-phosphate.
Straight chain of glucose has an aldehyde on carbon 1 whereas the fructose has a ketone on carbon 2.
Hence, the isomerisation is a conversion between an aldose to a ketose.
Catalyzed by phosphoglucose isomerase –> takes several steps as it has to open the ring structure in order to catalyze the reaction.
Explain the second phosphorylation reaction in glycolysis? (Step 3)
Fructose-6-phosphate is phosphorylated using ATP to form Fructose-1,6-bisphosphate.
This reaction is catalyzed by phosphofructokinase (PFK), an allosteric enzyme that sets the pace of glycolysis.
Explain what happens in the first cleavage reaction in glycolysis? (Step 4)
Fructose 1,6-bisphosphate is cleaved into glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP), completing stage 1 of glycolysis.
This reaction, which is readily reversible, is catalyzed by aldolase –> enzyme derives its name from the nature of the reverse reaction, an aldol condensation.
Describe the isomerisation reaction of DAHP in glycolysis? (Step 5)
Triosephosphate isomerase catalyzes the isomerization of DAHP into GAP.
This has to be done as DAHP cannot continue in the glycolytic pathway –> if not converted ATP will be lost.
The reaction is rapid and reversible –> normally at equilibrium there is 96% of DAHP –> but subsequent reactions in glycolysis remove the product –> driving the reaction to the RHS.
What happens to the two GAP molecules in glycolysis? (Step 6)
Conversion of glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate (1,3-BPG), a reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase.
Sum of two processes: the oxidation of the aldehyde to a carboxylic acid by NAD+ and the joining of the carboxylic acid and orthophosphate to form the acyl-phosphate product.
This creates a molecule with a high phosphoryl-transfer potential.
Describe what happens to the two 1,3-biphosphoglycerate molecules in glycolysis. (Step 7)
1,3-Bisphosphoglycerate is an energy-rich molecule with a greater phosphoryl-transfer potential than that of ATP. Thus, 1,3-BPG can be used to power the synthesis of ATP from ADP.
Phosphoglycerate kinase catalyzes the transfer of the phosphoryl group from the acyl phosphate of 1,3- bisphosphoglycerate to ADP.
The formation of ATP in this manner is referred to as substrate-level phosphorylation because of the phosphate donor.
Background info –> energy released of the oxidation of glyceraldehyde-3- phosphate to 3-phosphoglycerate is temporarily trapped as 1,3-bisphosphoglycerate –> energy powers the transfer of a phosphoryl group from 1,3-bisphosphoglycerate to ADP to yield ATP.
What happens to 3-phosphoglycerate in glycolysis? (Step 8)
Rearrangement reaction –> The position of the phosphoryl group shifts in the conversion of 3-phosphoglycerate into 2-phosphoglycerate, a reaction catalyzed by phosphoglycerate mutase.
Generally –> Mutase is an enzyme catalyzes the intramolecular shift of a chemical group, such as a phosphoryl group.
What happens to 2-phosphoglycerate in glycolysis? (Step 9)
Dehydration of 2-phosphoglycerate introduces a double bond, creating an enol.
Enolase catalyzes this formation of the enol phosphate phosphoenolpyruvate (PEP).
Dehydration reaction increases the transfer potential of the phosphoryl group –> because the phosphoryl group traps the molecule in its unstable enol form –> when the phosphoryl group has been lost –> molecule forms the more stable ketone (pyruvate).
Hence, the difference in stability between the enol and the ketone form creates the high phosphoryl transfer potential.
How does fructose enter the glycolytic pathway?
- A major area of fructose metabolism is the liver –> Fructose is phosphorylated –> fructose to fructose 1-phosphate by fructokinase. Fructose 1-phosphate is then split into glyceraldehyde and dihydroxyacetone phosphate an intermediate in glycolysis.
- In other tissues, such as adipose tissue –> fructose can be phosphorylated to fructose 6-phosphate by hexokinase –> enter the glycolytic pathway.
How does galactose enter the glycolytic pathway?
Galactose is converted into glucose 6-phosphate in four steps.
- Galactose to galactose-6-phosphate –> galactokinase.
- Galactose-6-phosphate then attains a uridyl group from uridine diphosphate glucose (UDP-glucose) –> Enzyme: galactose 1-phosphate uridyl transferase. Products: UDP-galactose and glucose 1-phosphate
- Enzyme UDP-Galactose-4-epimerase –> galactose moiety of UDP-galactose is then epimerized to glucose
- Finally, glucose 1-phosphate, formed from galactose, is isomerized to glucose 6-phosphate by phosphoglucomutase.
Describe the first step in the citric acid cycle –> condensation of OAA to citrate.
Condensation of a four-carbon unit, oxaloacetate, and a two-carbon unit, the acetyl group of acetyl CoA. Oxaloacetate reacts with acetyl CoA and H2O to yield citrate and CoA.
The first step is an aldol reaction (produces citryl CoA –> energy-rich due to thioester bond) which is followed by a hydrolysis reaction (driving reaction –> pushes reaction forward) –> both catalyzed by citrate synthase.
Describe the second step of the citric acid cycle –> citrate to isocitrate.
Hydroxyl group is not properly situated for oxidative decarboxylation in the next step –> citrate has to undergo isomerization.
Isomerization of citrate is accomplished by a dehydration step followed by a hydration step. The result is an interchange of an H+ and an OH_.
The enzyme catalyzing both steps is called aconitase because cis-aconitate is an intermediate.
Describe the third step of the citric acid cycle –> isocitrate to alpha-ketoglutarate.
Oxidative decarboxylation
First of four oxidation-reduction reactions in the citric acid cycle.
The oxidative decarboxylation of isocitrate is catalyzed by isocitrate dehydrogenase.
- Oxidation –> Isocitrate to Oxalosuccinate
- Decarboxylation –> Oxalosuccinate to alpha-ketoglutarate
The intermediate in this reaction is oxalosuccinate, an unstable beta-ketoacid. While bound to the enzyme, it loses CO2 to form alpha-ketoglutarate.
Produces first high-transfer-potential electron carrier –> NADH.
Describe the fourth step of the citric acid cycle –> alpha-ketoglutarate to Succinyl CoA
Oxidative decarboxylation reaction, the formation of succinyl CoA from alpha-ketoglutarate.
Catalyzed by the a-ketoglutarate dehydrogenase complex –> an organized assembly of three kinds of enzymes
Describe the fifth step of the citric acid cycle –> Succinyl CoA –> succinate.
Cleavage reaction of the thioester bond –> removes CoA
Coupled with the phosphorylation of a purine nucleoside diphosphate, usually ADP.
This reaction, which is readily reversible, is catalyzed by succinyl CoA synthetase (succinate thiokinase).
Note –> ADP or GDP can be used for the phosphorylation
Depends on the needs of the cells/tissue –> large amounts of cellular respiration, (skeletal and heart muscle) the ADP-requiring isozyme predominates. In tissues that perform many anabolic reactions, such as the liver, the GDP-requiring enzyme is common.
Note –> nucleoside diphosphokinase enzyme can be used to interconvert between GTP and ATP.
Describe the fifth step of the citric acid cycle –> succinate –> fumarate.
Succinate is oxidized to fumarate by succinate dehydrogenase. The hydrogen acceptor is FAD rather than NAD+.
FAD is the hydrogen acceptor in this reaction because the free-energy change is insufficient to reduce NAD+ .
Describe the sixth step of the citric acid cycle –> fumarate –> L-Malate.
Hydration of fumarate to form L-malate. Fumarase catalyzes a stereospecific trans addition of H+ and OH_ . The OH group adds to only one side of the double bond of fumarate; hence, only the L isomer of malate is formed.
Draw the entire citric acid cycle.
What is the second control point in the citric acid cycle?
A second control site in the citric acid cycle is alpha-ketoglutarate dehydrogenase, which catalyzes the rate-limiting step in the citric acid cycle.
Alpha-Ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH, the products of the reaction that it catalyzes. In addition, alpha-ketoglutarate dehydrogenase is inhibited by a high energy charge. Thus, the rate of the cycle is reduced when the cell has a high level of ATP
Explain the structure of Ubiquinone.
Coenzyme Q is a quinone derivative with a long tail consisting of five carbon isoprene units that account for its hydrophobic nature –> number of isoprene units differs on the species –> mammalian form contains 10 isoprene units (coenzyme Q10)
It may exist in several oxidation states.
- Fully oxidized –> two keto groups
- Oxidation of one electron and one proton –> semiquinone (QH)
- Semiquinone can lose a proton to form a semiquinone radical anion (Q .-)
- Fully reduced state –> 2 e- and 2 H+ –> ubiquinol (QH2)
What are the different iron-sulfur clusters found in biological systems?
Iron–sulfur clusters or iron–sulfur proteins (also called nonheme iron proteins) play a critical role in a wide range of reduction reactions in biological systems.
Several different types are known:
- Single iron ion is tetrahedrally coordinated to the sulfhydryl groups of four cysteine residues of the protein.
- Second kind, denoted by 2Fe-2S, contains two iron ions, two inorganic sulfides, and usually four cysteine residues.
- Third type, designated 4Fe-4S, contains four iron ions, four inorganic sulfides, and four cysteine residues
What happens to the electrons from NADH when it gets oxidised at complex 1?
- Binding of NADH –> transfer of its two high potential electrons to the flavin mononucleotide (FMN) prosthetic group –> creates reduced form FMNH2 .
Note –> The electron acceptor of FMN, the isoalloxazine ring, is identical with that of FAD.
- Electrons are then transferred from FMNH2 to a series of iron–sulfur clusters the second type of prosthetic group in complex I. ]
- Eventually, these electrons get used to reduce Coenzyme Q –> QH2 .
Explain the structure of complex I in the E.T.C.
Protein is L-shaped –> region sticking out into the matrix (hydrophilic) and an inner-membrane region (hydrophobic)
Membrane region
Has four proton half-channels consisting, in part, of vertical helices –> One set of half channels are on the matrix side whereas the others are on the inner membrane space side.
The Vertical helices on the matrix side are linked by a long horizontal helix –> connects the matrix half channels.
The cytoplasmic half-channels are joined by a series of beta-hair-pin-helix connecting elements (BH).
An enclosed Q-chamber –> site where Q accepts electrons exists between the junction of the hydrophilic and hydrophobic region of the protein
Finally, a hydrophilic A.A. funnel connects the Q chamber to a water-lined channel (into which the half channels open) that extends the entire length of the membrane-embedded portion.
Explain the Q-cycle in complex III.
QH2 passes two electrons to complex III but it only can accept one.
Overall –> Two QH2 molecules bind to the complex consecutively, each giving up two electrons and two H2 –> These Protons are released on the cytoplasmic side of the membrane.
- First QH2 binds to first Q binding site (Qo) –> releases it electrons which travel through the compelx to different destinations.
- One electron –> 2Fe-2S cluster –> cytochrome c1 –> then to Cytochrome C turning it into its reduced form. –> free to diffuse to complex IV.
- The second electron passes through two heme groups (low to high affinity) –> used to reduce oxidized ubiquinone in second binding site to form semiquinone radical anion. - Now fully oxidized Q in first binding site leaves to enter Q pool.
- A second molecule of QH2 binds to the Qo of complex III and reacts in the same way as the first –> one e- to Cyto C whereas, the other is used to reduce Q radical to an anion (second binding site).
- On addition of the second electron, the anion takes up two protons from the matrix side –> The removal of these two protons from the matrix contributes to the formation of the proton gradient
As a whole –> Four protons are released on the cytoplasmic side, and two protons are removed from the mitochondrial matrix.
Explain how electrons from cytochrome C are used to convert O2 into H2O.
- 2 Cytochrome C is oxidized and releases two electrons –> they flow down an electron-transfer pathway within cytochrome c oxidase, one stopping at CuB and the other at heme a3 .
- Both centres are in the reduced state so they can now bind to an oxygen molecule.
- When it binds the O2 obtains the 2 electrons from the centres in order to form a peroxide bridge between CuB and Heme a3 .
- Two more molecules of cytochrome C are oxidised and the released electrons travel to the active center.
- Addition of two electrons plus 2 H+ is used to reduce the oxygens to -OH (forms CuB2+-OH and Fe3+-OH).
- Reaction with two more H+ ions allows the release of two molecules of H2O and resets the enzyme to its initial, fully oxidized form.
Note –> the four protons come from the matrix –> directly contribute to proton gradient.
Also note that during this entire process the complex transports another 4 protons into the inner-membrane space –> total of 8 protons removed from matrix –> mechanism not clear.
How does the cell deal with reactive oxygen species (ROS)?
Superoxide dismutase –> This enzyme scavenges superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen.
The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen.
Explain the structure of ATPase.
Can be described as a ball on a stick.
Stick = F0 subunit –> inner mitochondrial membrane
Ball = F1 subunit –> produdes into matrix –> catalytic activity of ATPase.
F1 subunit –> hydrophilic segment
Five types of polypeptide chains (α3 , β3, γ, δ, ε) with the indicated stoichiometry
α and β subunit make up the bulk of the subunit –> form hexameric ring –> arranged alternately –> homologous to each other –> Both bind nucleotides but only the β subunits are catalytically active.
Below the α and β subunits is a central stalk consisting of the γ and ε proteins –> γ long helical coiled coil that extend into the center of the hexamer –> γ breaks the symmetry of the α and β subunits –> forms different interactions –> different conformation.
F0 subunit –> hydrophobic
F0 contains the proton channel of the complex
Channel –> a ring comprising from 8 to 14 c subunits that are embedded in the membrane.
A single a subunit binds to the outside of the ring.
General
The F0 and F1 subunits are connected in two ways: by the central stalk and by an exterior column. The exterior column consists of one a subunit, two b subunits, and the ε subunit.
What is the exact reaction catalyzed by the F1 subunit of ATP synthase?
ADP3- + HPO42- + H+ <—> ATP4- + H2O
Terminal oxygen atom of ADP attacks the Pi –> forms a pentacovalent intermediate –> dissociates to form ATP and H2O.
How many active sites are there on the F1 subunit? What are the different active sites?
Since there are 3 Beta subunits –> Beta subunits are the only sites that are catalytically active –> Hence, there are three active sites on the enzyme, each performing one of three different functions at any instant.
At any one moment…
Loose active site –> This conformation ADP + Pi bind (note –> can not release once bound)
Tight active site –> Bind to ATP with high avidity –> it converts ADP + Pi into ATP. (note –> can not release once bound)
Open active site –> has a more open conformation and can bind or release adenine nucleotides.
Note –> rotation of the γ subunit by 120 degrees –> changes the conformation of the Beta subunits –> thus also changing their function (L, T or O).
Explain the movement of protons through the Fo subunit.
- The cytoplasmic half channel is deprotonated.
- Proton from the Intermembrane space enters the half channel and protonates the glutamate residue.
- The ring structure rotates clockwise –> movement of protons through the half channels from the high proton concentration of the cytoplasm to the low proton concentration of the matrix powers the rotation of the c ring.
Note –> a subunit remains stationary throughout the entire process.
- The protonated glutamic acid residue is now in the matrix half-channel.
- Glutamic acid gets deprotonated –> releases H+ into the matrix.
How do electrons from cytoplasmic NADH (i.e. glycolysis) enter the mitochondria?
The inner mitochondrial membrane is impermeable to NAD+ and NADH.
Solution –> transport electrons from NADH not NADH itself –> Glycerate 3-phosphate shuttle.
- Transfer of a pair of electrons from NADH to dihydroxyacetone phosphate –> to form glycerol 3-phosphate –> catalyzed by a glycerol 3-phosphate dehydrogenase in cytoplasm
- Glycerol 3-phosphate dehydrogenase moves into the inner-membrane space of the mitochondria.
- Reoxidized back to dihydroxyacetone phosphate –> catalyzed by an enzyme that is on the surface of the inner mitochondrial membrane (glycerol 3-phosphate dehydrogenase).
- Electron pair from glycerol-3-phosphate is transferred to a FAD prosthetic group on the enzyme -> converted to FADH2.
- Electrons then used to reduce Q to form QH2 .
Note –> 1.5 ATP is produced rather than the expected 2.5 from NADH because FAD is the electron acceptor.
This method of electron transfer is predominant in muscles –> allows the cell to maintain high levels of oxidative phosphorylation.
How many ATP molecules are generated by the complete oxidation of glucose to CO2?
About 30 molecules of ATP are formed when glucose is completely oxidized to CO2 .
The calculation showed below.
