Biochemistry
Arash Azarfar
Lorestan University Biochemistry
• Principle of energy release from food
• Release of energy in the form of ATP • Glucose, fat and amino acids
Lorestan University Biochemistry
• Principle of energy release from food
• Biological oxidation and H-transfer systems
• Oxidation does not necessary involve oxygen:
• May be involve in only electron removal
• Ferrous/Ferric system
Lorestan University Biochemistry
• Principle of energy release from food
• Biological oxidation and H-transfer systems
• Oxidation does not necessary involve oxygen:
• The removal of e- accompanied by protons from a hydrogenated molecule
Lorestan University Biochemistry
• Principle of energy release from food
•The removal of e- accompanied by protons from a hydrogenated molecule • Electron transfers from a donor to an acceptor • Electron transferred: • Accompanied by proton • Proton may be liberated into solution
Lorestan University Biochemistry
• Principle of energy release from food
•The ultimate electron acceptor in the aerobic cell
• Oxygen
Lorestan University Biochemistry
• Principle of energy release from food
•Oxygen is only the ultimate electron acceptor in the cell • Other electron acceptors form a chain
Lorestan University Biochemistry
• Principle of energy release from food
•Other electron acceptors form a chain • Electron transport chain • Plays a predominant role in ATP generation
Lorestan University Biochemistry
Lorestan University Biochemistry
• Principle of energy release from food
•NAD+ (Nicotinamide adenine dinuclotide) • Involved in oxidation of many metabolites • General structure
Lorestan University Biochemistry
• Principle of energy release from food
•NAD+ (Nicotinamide adenine dinuclotide) • Involved in oxidation of many metabolites • General structure
Lorestan University Biochemistry
• Principle of energy release from food
•NAD+ (Nicotinamide adenine dinuclotide) • Is a coenzyme • Differs from an ordinary enzyme substrate
• Reduced compound leaves the enzyme and attaches to the second enzyme
Lorestan University Biochemistry
•NAD+ (Nicotinamide adenine dinuclotide)
• Business end
Nicotinamide
Lorestan University Biochemistry
• Accepting 2 e- plus one proton as hydride ion (H:-)
• The second proton being liberated into solution
Lorestan University Biochemistry
•NAD+ (Nicotinamide adenine dinuclotide)
• Coenzyme for several dehydrogenases
Lorestan University Biochemistry
• FAD and FMN
• FAD (flavin adenine dinucleotide) • Derived from Riboflavin • Important feature:
• Accepting two H atoms to become FADH2 (in combination with appropriate proteins)
• FAD is a prosthetic group (permanent attachment to its apoenzyme)
Lorestan University Biochemistry
• FAD and FMN • Structure
Lorestan University Biochemistry
• Energy release from glucose • Glucose or glycogen
• The main stages of glucose oxidation
• ΔG0´ is -2820 kJ mol-1 • Oxidation is accompanied with more than30 mol ATP
Lorestan University Biochemistry
• Oxidation of glucose:
• Glycolysis
• Splitting the glucose into C3 fragments • Citric acid cycle (tricarboxylic acid, TCA; Krebs)
Pyruvate C Acetyl group electron electron carriers
• No oxygen is involved
• Carbon atoms are released as CO2
Lorestan University Biochemistry
• Oxidation of glucose:
• Electron transport system • Transporting of electrons from electron carriers to oxygen where, with protons form water • Occurs in inner membrane of mitochondrial
membrane (eukaryote cells)
Lorestan University Biochemistry
• Oxidation of glucose:
• Glycolysis • Does not involve oxygen • Production of 2 mol ATP • End products: Pyruvate and NADH
Lorestan University Biochemistry
• Oxidation of glucose:
• Glycolysis • Does not involve oxygen • Production of 2 mol ATP • End products: Pyruvate and NADH
Lorestan University Biochemistry
• Oxidation of glucose:
• Glycolysis • Aerobic glycolysis
• Reoxidizing the NADH via mitochondria • Taking up the pyruvate by mitochondria
Lorestan University Biochemistry
• Oxidation of glucose:
• Glycolysis • Anaerobic glycolysis • No NAD+ no glycolysis • At high glycolytic rate/or inadequate oxygen glycolysis needed to generate ATP • Emergency system comes to play; reducing the pyruvate to lactate
Lorestan University Biochemistry
• Oxidation of glucose:
• Glycolysis • Anaerobic glycolysis • There is a lot of lactate dehydrogenase in muscles: • NADH can be rapidly reoxidized (permission of ATP synthesis) • Permits glycolysis to proceed at very fast rate • Vast amount of glucose can be broken down • Produced lactate leaks out; in the liver will be converted into glucose
Lorestan University Biochemistry
• Oxidation of glucose:
• Glycolysis • Anaerobic glycolysis • Yeast can live entirely on anaerobic glycolysis
Lorestan University Biochemistry
• Oxidation of glucose:
• Glycolysis • Anaerobic glycolysis • Yeast can live entirely on anaerobic glycolysis
Does not occur in animals
Lorestan University Biochemistry
• Oxidation of glucose:
• Citric acid cycle • The main mean energy-generating units of aerobic cells
The site of ATP generation
Stage 2 of glucose metabolism mainly occurs
Lorestan University Biochemistry
• Oxidation of glucose:
• Citric acid cycle • Aerobic glycolysis produces: • Pyruvate and NADH in the cytoplasm • To be further oxidized pyruvate must enter the mitochondria • NADH is oxidizes via mitochondria: • Electrons are transported in and oxidized, leaving NAD+ in cytoplasm
Note: some cells such as erythrocytes have no mitochondria; ATP must generate by glycolysis; they are glucose dependent
Lorestan University Biochemistry
• Oxidation of glucose:
• Citric acid cycle • How is pyruvate fed into the TCA
• Acetyl-CoA • What is CoA? (CoA-SH) • CoA unlike NAD+ and FAD is not an electron carrier • CoA is an acyl group carrier • Contains pantothenic acid
Lorestan University Biochemistry
• Oxidation of glucose:
• Citric acid cycle • How is pyruvate fed into the TCA •Contains pantothenic acid
Lorestan University Biochemistry
• Oxidation of glucose:
• Citric acid cycle •Contains pantothenic acid: • It is just there and apparently quite inert • Provides a recognition group
The business end of molecule
Lorestan University Biochemistry
• Oxidation of glucose:
• Citric acid cycle
• The CoA molecule carries acyl group
as thiol esters; CH3CO—S—CoA -1 • ΔG0 thiol ester is – 31 kJ mol vs. – 20 kJ mol-1 carboxylic ester
Lorestan University Biochemistry
• Oxidation of glucose:
• Oxidation decarboxylation of pyruvate:
• CO2 is released (decarboxylation) • A pair of electron is transferred to NAD+ (oxidation) • An acetyl group is transferred to CoA • The reaction is irreversible Pyruvate dehydrogenase
Lorestan University Biochemistry
• Oxidation of glucose:
• Three molecule of CO2 + • Three molecule of NAD are reduced • One molecule of FAD are reduced
Lorestan University Biochemistry
• Oxidation of glucose • Electron transport to oxygen
• Oxidation of NADH and FADH2 take place in inner membrane of mitochondria • The electron transport chain- a hierarchy of electron carriers • Redox potentials: • Problem: transference of electrons from NADH and
FADH2 to oxygen
Lorestan University Biochemistry
• Oxidation of glucose •Redox potentials: • Problem: transference of electrons from NADH and
FADH2 to oxygen
ΔG0´=-220 kJ mol-1
Lorestan University Biochemistry
• Oxidation of glucose •Redox potentials: • Problem: transference of electrons from NADH and
FADH2 to oxygen • Redox potential: • Compounds are capable of being oxidised (electron donors) • Oxido-reduction reactions: • Electron donor (reductant); electron acceptor (oxidant)
Lorestan University Biochemistry
• Oxidation of glucose •Oxido-reduction reactions: • Electron donor (reductant); electron acceptor (oxidant)
Redox couple
•X and X-; Y and Y- are such couples
Lorestan University Biochemistry
• Oxidation of glucose •Oxido-reduction reactions: • Different redox couples have different affinities for e- • Lesser affinity means tending to donate electron to another of high affinity
´ • Redox potential (E0 ): • Is a measurement of electron affinity or electron donating of a redox couple
Lorestan University Biochemistry
• Oxidation of glucose
´ •Redox potential (E0 ) • Importance in biochemical reactions: • Indicator of the direction in which electrons will tend to flow • Important:
´ • Relationship between E0 and free energy changes
Lorestan University Biochemistry
•Nernest equation
• n =number of electrons transferred in the reaction • F= Faraday constant (96.5 kJ V-1 mol-1)
Lorestan University Biochemistry
• Oxidation of glucose •Electrons are transported in a stepwise fashion • In the cell, transport of electrons to oxygen does not happen in a single step • Chain of electron carriers:
Carriers of ever-increasing redox values (decreasing reducing potential)
Terminating to the ultimate electron acceptor, oxygen
Lorestan University Biochemistry
• Oxidation of glucose •Electrons are transported in a stepwise fashion
• NADH and FADH2 bump down a staircase
Each step being a carrier of the appropriate redox potential and each fall releasing free energy
Lorestan University Biochemistry
• Oxidation of glucose
Harnessing the energy (indirectly results in ATP generation rather than being wasted as heat) Liberation of free energy in manageable parcels
Lorestan University Biochemistry
• Oxidation of glucose
• The oxidation of NADH and FADH2 drives the conversion of ADP and Pi to ATP • The complete process is called oxidative phosphorylation: • 30 mol ATP per mol oxidised ATP
Lorestan University Biochemistry
Lorestan University Biochemistry
• Energy release from oxidation of fat • Oxidized fat can also provide energy for ATP production • In terms of energy production: • Fatty acid components of TAG are quantitatively important • Glycerol portion being less important • Fatty acids are quite different from glucose • Expectation: their oxidation differ from each other
• Note: Metabolism of different foodstuffs dovetails together with a majestic simplicity
Lorestan University Biochemistry
• Energy release from oxidation of fat
• Glucose oxidised, acetly-CoA is formed and fed into TCA • Fatty acids are also so manipulated: • Carbon atoms are detached two at a time as acetly-CoA
Fed into TCA • Fatty acids oxidation differ from that of glucose only in preliminary formation of TCA
Lorestan University Biochemistry
• Energy release from oxidation of fat
• In the preliminary manipulation:
NAD+ and FAD are reduced
e-
Electron transport chain
Lorestan University Biochemistry
• Energy release from oxidation of amino acids • There are 20 amino acids: • Presence in excess of immediate requirements: • Removing their amino group • Using their carbon-hydrogen skeleton as fuel:
• Converted to pyruvate, acetly-CoA or intermediates of TCA
Lorestan University Biochemistry
•Using their carbon-hydrogen skeleton as fuel:
• Converted to pyruvate, acetly-CoA or intermediates of TCA •
• TCA thus plays an important role in metabolism
Lorestan University Biochemistry
• Only two are solely ketogenic: • Lysine and Leucine •Biochemistry.ppt#162. Slide 162
Lorestan University Biochemistry
• The interconvertibility of fuels: • Glucose in excesses can be converted to fat
• In animals fatty acids cannot be converted to glucose: • Acetyl-CoA cannot be converted to pyruvate: • Pyruvate dehydrogenase reaction is irreversible
Lorestan University Biochemistry
• In animals fatty acids cannot be converted to glucose: • Acetyl-CoA cannot be converted to pyruvate: • Pyruvate dehydrogenase reaction is irreversible
• Bacteria and plants have the capacity to convert fats and C2 compounds such as acetate to glucose
Lorestan University Biochemistry
•Bacteria and plants have the capacity to convert fats
and C2 compounds such as acetate to glucose:
• Glyoxylate pathway: • A route involving enzymes of both the mitochondrial and glyoxyzome •Biochemistry.ppt#167. Slide 167
Lorestan University Biochemistry
•Glyoxylate pathway:
• Results: • Net conversion of acetyl-CoA to glyoxylate instead of
to two molecules of CO2 as occurs in TCA 2 Acetyl-CoA+2 NAD++FAD oxaloacetate+2 CoA+2NADH+FADH+2H+
Lorestan University Biochemistry
•Thermodynamic: Principles •Living things require a continuous throughput of energy • Conversion of radiant energy to the chemical energy (stored as chemical substances) by plant • Metabolizing the substances by plant or animal to power:
• Synthesis of biomolecules • Maintenance of concentration gradients • Movement of muscles • These processes ultimately transform the energy to heat
Lorestan University Biochemistry
•Thermodynamic: Principles • A considerable portion of the cellular biochemical apparatus: • Must be devoted to the acquisition and utilisation of energy
• Thermodynamic (therme, heat + dynamics, power)
• Description of the relationships among the various forms of energy • How energy affects matter on the macroscopic levels as apposed to the molecular level
Lorestan University Biochemistry
• Thermodynamic (therme, heat + dynamics, power) • Description of the relationships among the various forms of energy • How energy affects matter on the macroscopic levels as apposed to the molecular level : • Deals with the amounts of matter large enough for their average properties: • Temperature • Pressure to be well defined
Lorestan University Biochemistry
• Thermodynamic (therme, heat + dynamics, power) • The basic principles were developed in nineteenth century • Before acceptance of atomic theory • With knowledge of thermodynamics: • Determine whether a physical process is possible
Lorestan University Biochemistry
• Thermodynamic (therme, heat + dynamics, power) • Necessary for understanding :
• Why macromolecules fold to their native conformations • How metabolic pathways are designed • Why molecules cross biological membranes • How muscle generate mechanical forces and so on
Lorestan University Biochemistry
• Thermodynamic (therme, heat + dynamics, power) • Note: • Thermodynamics does not indicate the rates at which possible processes actually occur: • Glucose and oxygen reaction:
• Thermodynamics tells us this reaction will happen with releasing the copious amount of energy • Yet, it does not indicate that the mixture is indefinitely stable at room temp (in the absence of the appropriate enzymes)
Lorestan University Biochemistry
• Thermodynamic (therme, heat + dynamics, power) • Prediction of reaction rates requires mechanistic description of molecular process: • Yet, mechanistic models must conform to thermodynamics principles
Lorestan University Biochemistry
• Thermodynamic (therme, heat + dynamics, power) •In biochemistry is most frequently concerned with: • Conditions under which processes occur spontaneously : • First and second laws of thermodynamics • The concept of free energy • Nature of processes at equilibrium
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED • A system: A part of universe that is of the interest; a reaction vessel or an organism • Surrounding: the rest of the universe • System: Open or close • Depending on whether or not it can exchange matter and energy with its surrounding : • Example: living organism
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED • Energy: • First law of thermodynamics is a mathematical statement of the law of conservation of energy: • Energy can be neither created nor destroyed
ΔU = Ufinal - Uinitial = q – w (1) q = The heat absorbed by the system from the surrounding w = The work done by the system on the surrounding
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED •Heat: • Reflection of random molecular motion • Work: Force times the distance moved under its influence • Associated with organised motion
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED •Heat: • Processes in which system releases heat (negative q) are known as exothermic processes (exo, out of) • Those in which system gain heat (positive q) are known as endothermic processes (endo, within) • Work: • Under this convention, work done by the system against an external force is defined as a positive quantity
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED •State Functions Are Independent of the Path a System Follows • Energy of a system depends only on: • Its current properties or state, not on how it reached that state • Example: The state of a system composed of a particular gas • Can be completely described by its pressure and temperature (State functions)
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED •State Functions Are Independent of the Path a System Follows • The energy of this gas sample is a function only of these so-called state functions (quantities that depend only on the state of the system) • Consequently: • ΔU = 0 for any process in which the system returns to its initial state (cyclic process)
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED •State Functions Are Independent of the Path a System Follows • Neither heat nor work is separately a state function: • Each is dependent on the path followed by a system in changing from one state to another •Initial state Finial stage Gas Do work against an external pressure or do no work by following path in which it encounters no external resistance
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED
•Initial state Finial stage Gas
Do work against an external pressure or do no work by following path in which it encounters no external resistance
Work is path dependent
Example: Your bank account, neither Δq nor Δw
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED •Enthalpy • Any combination of only state functions must also be a state function: • Enthalpy (enthalpein, to warm in)
H = U + PV (2) V = volume of the system P = Pressure of the system
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED •Enthalpy (enthalpein, to warm in) • Is a convenient quantity with which to describe biological system: • Under constant pressure (Typical of most biological system) • The enthalpy change between the initial and final states of process (ΔH): • Heat that it generates or absorbs (can be easily measured)
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED •ΔH, Heat that it generates or absorbs (can be easily measured) • Dividing work into two categories: • Pressure-volume work (work is performed by expansion against an external pressure, PΔV ) • all other works (w´): w = PΔV + w´ (3) Combining the equations 2 and 3
ΔH = ΔU + PΔV = qp – w + PΔV = qp - w´
Lorestan University Biochemistry
• First Law of thermodynamics: ENERGY IS CONSERVED •ΔH, Heat that it generates or absorbs (can be easily measured)
ΔH = ΔU + PΔV = qp – w + PΔV = qp - w´
qp =heat transferred at constant pressure, so if w´ = 0 which is often true of chemical reactions
ΔH = qp
• The volume changes in most biochemical processes are negligible, so: • The differences between ΔU and ΔH values are usually insignificant
Lorestan University Biochemistry
• Understanding the utility of state functions •Enthalpy change resulting from:
• Complete oxidation of 1 g glucose to CO2 and H2O by muscle tissues
• Directly would present enormous experimental difficulties: • Interference of metabolic reactions not involving in glucose oxidation with enthalpy measurements
• Enthalpy is a state function
Lorestan University Biochemistry
• Understanding the utility of state functions
• Enthalpy is a state function • Measuring glucose’s enthalpy of combustion in any apparatus of our choosing (constant pressure calorimeter rather than muscle), yes or no? • Yes as long as we can establish the final metabolic products
(CO2 and H2O) • Same reactants and same products, no matter of reaction pathway
Lorestan University Biochemistry
• Second Law of Thermodynamics: The Universe Tends Towards Maximum Disorder
• Example: A swimmer falls into the water (Spontaneous) • Conversion of his coherent body motion energy to that of chaotic thermal motion of the water • The reverse motion has been never witnessed • Spontaneous Process: • Conversion of order (coherent motion of swimmers body) to chaos (random thermal motion of the water molecules)
Lorestan University Biochemistry
• Second Law of Thermodynamics: The Universe Tends Towards Maximum Disorder • Provides a criterion for spontaneity of a process
Lorestan University Biochemistry
• Second Law of Thermodynamics: The Universe Tends Towards Maximum Disorder • Spontaneity and disorder: • The spontaneous processes occur in directions that increase the overall disorder of the universe
•Disorder: The number of equivalent ways, W, of arranging the components of the universe • Example: Isolated system consisting of two bulbs
Lorestan University Biochemistry
•Example: Isolated system consisting of two bulbs of equal volume containing a total N identical molecules of ideal gas
• 2N= Equally probable ways that the N molecules distribute among the two bulbs • Gas molecules are indistinguishable; N + 1 states of the system: 0, 1 ,…, (N -1), N
Equilibrium
Lorestan University Biochemistry
•Example: Isolated system consisting of two bulbs of equal volume containing a total N identical molecules of ideal gas
• The number of ways, WL, of placing L of the N molecules in the left bulb: N! W L L!(N L)!
• The probability that such state N occurs: W L/2
Lorestan University Biochemistry
•For any N, the most probable state is the one with the highest
value of WL • Half of molecules in one bulb (L = N/2 for N even) • N = 10, the probability that L is within 20 % 0f N/2 (4, 5 or 6) is 0.66 • N = 50, this probability (L is in the range of 20-30) is 0.88 • N= 10 23 , the number of molecules in the right bulb differs from that of the left is 1 in every 10 billion
• As N increases, the probability that L is nearly equal to N/2 approaches unity
Lorestan University Biochemistry
•The reason for equality of gas molecules in each bulb is not because of :
• Any law of motion; the energy of the system is the same for any arrangements of the molecules • It is because the aggregate probability of all other states is so utterly insignificant
Lorestan University Biochemistry
9! W 126 4 4!(9 4)!
Lorestan University Biochemistry
•Entropy
• In chemical system, W:
• The number of equivalent ways of arranging system in a particular state
• Usually inconveniently immense, •Example: • The same twin bulb system, N gas molecule
N ln2 WN / 2 10
Lorestan University Biochemistry
•Entropy •Example: • The same twin bulb system, N gas molecule N ln2 WN / 2 10
• N = 23
71022 W 22 10 510
Lorestan University Biochemistry
•Entropy
• To deal with W more easily Ludwig Boltzmann (1877) defined a quantity, Entropy
S kB lnW
-23 -1 • kB = 1.3807 × 10 J K • For the twin bulbs system:
S kB N ln 2
Lorestan University Biochemistry
•Entropy
• The law of random chance cause: • Any system of reasonable size to spontaneously adopt its most probable arrangement • The one in which the entropy is maximum • This state is so overwhelmingly probable • Slide 83 •Entropy.xls
Lorestan University Biochemistry
•Entropy
• Spontaneous process causes the system entropy’s to increase • For any constant energy process (ΔU = 0), a spontaneous process is characterized by ΔS > 0
• Since energy of the universe is constant (first law of thermodynamics): • Any spontaneous process must cause the entropy of the universe to increase
Lorestan University Biochemistry
•Entropy
•Since energy of the universe is constant (first law of thermodynamics): • Any spontaneous process must cause the entropy of the universe to increase
Ssystem Ssurrounding Suniverse 0 •General expression for the second law of thermodynamics: • Statement of the general tendency of all spontaneous processes to disorder the universe • In other words, the entropy of the universe tends towards a maximum
Lorestan University Biochemistry
•Entropy •Application of the second law of thermodynamics
• Why blood transports O2 and CO2 between the lungs and the tissues? • Solutes in solution behave analogously to gases: • Tendency to maintain a uniform concentration throughout their occupied volume • Their most probable arrangement
Lorestan University Biochemistry
Ssystem Ssurrounding Suniverse 0
•Does not imply that a particular system cannot increase its degree of order • A system can be ordered at the expense of disordering its surrounding • Application of energy to the system
Lorestan University Biochemistry
•A system can be ordered at the expense of disordering its surrounding • Application of energy to the system, • Example: living organisms • Well ordered, organised from the molecular levels upwards • Achieve this order at the expense of disordering the nutrients they consume
Lorestan University Biochemistry
•Measurement of entropy • It is impractical to determine it by counting the number of ways, W, in chemical and biological system • For the constant temperature, typical of biological processes q S T
T is the absolute temperature at which the change in heat occurs
Lorestan University Biochemistry
•Measurement of entropy •For the constant temperature, typical of biological processes q S • The equality = System remainsT in equilibrium throughout the change, reversible process • Entropy in such systems can be measured straightforwardly: • Measurements the heat transferred and the temperature at which this occurs
Lorestan University Biochemistry
• FREE ENERGY: THE INDICATOR OF THE SPONTANIETY • The spontaneity of a process cannot be predicted from the system entropy change alone
• Exothermic process (ΔHsystem < 0) may be spontaneous even though they have ΔSsystem < 0 • Example: Spark 2 mol H2 + 1 mol O2 2 mol H2O • Similarly many denatured proteins will spontaneously fold to their highly ordered native conformations
Lorestan University Biochemistry
• FREE ENERGY: THE INDICATOR OF THE SPONTANIETY • What we need; a state function: • To predict whether or not a process is spontaneous
• Gibbs Free Energy (1878) G = H – TS For systems that can only do pressure-volume work (w´= 0)
ΔG = ΔH – T ΔS = qp - T ΔS
Lorestan University Biochemistry
•Gibbs Free Energy (1878) G = H – TS For systems that can only do pressure-volume work (w´= 0)
ΔG = ΔH – T ΔS = qp - T ΔS In the spontaneous process at constant T T ΔS ≥ q Consequently ΔG ≤ 0 is the criterion of spontaniety
Lorestan University Biochemistry
•Gibbs Free Energy (1878)
•ΔG < 0 , spontaneous process, exergonic (ergon, work)
• ΔG > 0, not spontaneous process, endergonic
Note: the value of ΔG varies directly with temperature
Lorestan University Biochemistry
• Note: the value of ΔG varies directly with temperature ΔH ΔS ΔG = ΔH – T ΔS − + The reaction is both enthalpically (exothermic) and entropically favoured. It is spontaneous (exergonic) at all temperatures − − The reaction is enthalpically favoured but entropically opposed. It is spontaneous only at temperature below T= ΔH/ΔS + + The reaction is enthalpically opposed but entropically favoured. It is spontaneous only at temperature above T= ΔH/ΔS + − The reaction is both enthalpically (exothermic) and entropically oppsed. It is unspontaneous (endergonic) at all temperatures
Lorestan University Biochemistry
•Glycolysis, TCA, and the electron transport system:
• Glycolysis: • Splitting of glucose or a glycosyl unit of glycogen: • Two molecules of pyruvate • Reduction of NAD+ • Glucose or glycogen • In normal circumstances most tissues will have glycogen stores: • Glycolysis may be proceeding from glycogen rather than glucose
Lorestan University Biochemistry
•Glycolysis, TCA, and the electron transport system: •Glucose or glycogen • Producing the glucose-6-phosphate via glucose-1-phosphate • In the liver: hydrolyzing to release free glucose into blood • On the glycolytic pathway : Pyruvate • Free glucose obtained from the blood: • Conversion to G-6-P by phosphorylation using ATP
Lorestan University Biochemistry
•Glycolysis, TCA, and the electron transport system: • G-6-P: • Glycogen • Pyruvate • Blood glucose • Depends on how the metabolic control switches are set
Lorestan University Biochemistry
• The relationship between, No ATP glycolysis, glucose, and glycogen
• No ATP when starts with glycogen:
• The energy of glycosyl group (ΔG0´ of it hydrolysis) is similar to that of the phosphate ester: • Is preserved by phosphorolysis Mg2+
Why is needed?
Lorestan University Biochemistry
Lorestan University Biochemistry
•Why use ATP at the beginning of glycolysis • Glycolysis involves phosphorylated compounds • ATP must be used to phosphorylate glucose- It has the necessary energy potential • G-6-P is a low-energy phosphoryl compound • Certainly we have lost a high-energy phosphoryl group in using ATP • A 100% profit investment is made on The ATP used in glycolysis of ATP
Lorestan University Biochemistry
•Why use ATP at the beginning of glycolysis • Important question? • Why does hexokinase catalyze the transfer of a phosphoryl group to glucose to yield G6P, but not to water to yield ADP +
pi?
Lorestan University Biochemistry
•Why is G6P converted to F6P? • Conversion of G6P (aldose) to F6P (ketose) • The aim, is to split the molecule into two
C3 compounds • Aldol condensation • The reverse reaction, splitting an aldol into two parts- an aldhyde and a ketone
Lorestan University Biochemistry
•Why is G6P converted to F6P? • Conversion of G6P (aldose) to F6P (ketose) • G6P is not an aldol, but F6P is an aldol
• By forming the fructose isomer, the sugar phosphate can be
split into two C3 compounds by aldol reaction • The enzyme involved is phosphoglucose isomerase (PGI)
Lorestan University Biochemistry
Lorestan University Biochemistry
Lorestan University Biochemistry
• By forming the fructose isomer, the sugar phosphate can
be split into two C3 compounds by aldol reaction • The enzyme involved is phosphoglucose isomerase (PGI) • Before splitting, transferring another phosphoryl group from ATP
Lorestan University Biochemistry
• Splitting F1,6BP by the enzyme aldolase that catalyze the aldol reaction:
• Two C3 products having a phosphoryl group (how?)
Mg2+
Lorestan University Biochemistry
•Why is G6P converted to F6P? • Conversion of G6P (aldose) to F6P (ketose) • The aim, is to split the molecule into two
C3 compounds • Aldol condensation • The reverse reaction, splitting an aldol into two parts- an aldhyde and a ketone
Lorestan University Biochemistry
•Why is G6P converted to F6P? • Conversion of G6P (aldose) to F6P (ketose) • G6P is not an aldol, but F6P is an aldol
• By forming the fructose isomer, the sugar phosphate can be
split into two C3 compounds by aldol reaction • The enzyme involved is phosphoglucose isomerase (PGI)
Lorestan University Biochemistry
Lorestan University Biochemistry
• ΔG0´ for aldolase reaction is +23.8 kJ mol-1 which would seem to preclude its ready occurrence • The reaction is freely reversible (how)? • One molecule of reactants gives rise to two molecules of products: • ΔG of the reaction is influenced by concentration to an usual degree
Lorestan University Biochemistry
• ΔG0´ for aldolase reaction is +23.8 kJ mol-1 which would seem to preclude its ready occurrence • The reaction is freely reversible, while reactions with smaller ΔG0´ values will not proceed in the cell • ΔG0´ values are determined at 1 M concentrations of reactants and products • Concentrations in the cell are more likely to be at 10-3 to 10-4: • Actual ΔG values are always different from ΔG0´ values • ΔG0´ values are usually useful guides to metabolic events! This not true in the case of aldolase • The correlation between ΔG0´ values and ΔG values in the cell is very poor
Lorestan University Biochemistry
• ΔG0´ values are usually useful guides to metabolic events! This not true in the case of aldolase • The correlation between ΔG0´ values and ΔG values in the cell is very poor • One molecule of reactant, F-1:6DP GAP + DHAP
products G G RT ln reac tants that is,
GAPDHAP G G0 RT ln F 1: 6 BP
Lorestan University Biochemistry
•The correlation between ΔG0´ values and ΔG values in the cell is very poor • There are two products of low concentration • RT ln ([products]/[reactants]) has a large negative value • Giving a ΔG value compatible with ready reversibility in the cell • In rabbit skeletal muscle is – 1.3 kJ mol-1
Lorestan University Biochemistry
•Interconversion of GAP to DHAP by triose phosphate isomerase (TPI)
•Interconversion of GAP to DHAP
Lorestan University Biochemistry
•Interconversion of GAP to DHAP
Lorestan University Biochemistry
• Glyceraldehyde – 3- phoshapte dehydrogenase- an oxidation linked to ATP synthesis • Aldehyde group of GAP is oxidised by GAPDH, using NAD+ as electron acceptor • Expectation: producing a carboxyl group, but: • Oxidation of a – CHO group to – COO- has a large negative ΔG value: Sufficient to produce a high-energy
phosphate from Pi
Lorestan University Biochemistry
• Mechanism • At the active site, amino acid cysteine which has a thiol or sulphydryl group (-SH)
Oxidation by GAPDH
Lorestan University Biochemistry
• Mechanism • Thiol ester is a high-energy compound as that of phosphate compound
High-energy compound
Lorestan University Biochemistry
Lorestan University Biochemistry
• Mechanism • Thiol ester is a high-energy compound as that of phosphate compound
High-energy compound •Phosphoryl group can be transferred to ADP forming ATP
• Phosphoglycerate kinase
Lorestan University Biochemistry
•Phosphoryl group can be transferred to ADP forming ATP
• Phosphoglycerate kinase • The generated phosphoryl group in processed is attached to the substrate of an enzyme
• Substrate-level-phsphorylation
Lorestan University Biochemistry
•Substrate-level-phsphorylation
Lorestan University Biochemistry
•Substrate-level-phsphorylation
+ 0´ -1 GAP + Pi + NAD 1,3-BPG + NADH ΔG =+6.7 kJ.mol Mg2+ 1,3-BPG + ADP 3PG + ATP ΔG0´=- 18.8 kJ.mol-1
+ GAP + Pi + NAD + ADP 3PG + NADH + ATP ΔG0´=- 12.1 kJ.mol-1
Lorestan University Biochemistry
•The final step in glycolsis
Phosphoglycerate mutase • The reaction is necessary preparation for the next reaction in glycolysis
Lorestan University Biochemistry
•The final step in glycolsis Phosphoglycerate mutase • The reaction is necessary preparation for the next reaction in glycolysis: • Purpose: generation a high-energy phosphoryl compound for use in ATP synthesis • The reaction is not a really an intermolecular transfer of phosphoryl group
Lorestan University Biochemistry
Occasionally 2,3BPG leaves the enzyme
Lorestan University Biochemistry
•Occasionally 2,3BPG leaves the enzyme • Glycolysis influences oxygen transport • 2,3 BPG specifically binds to deoxyhemoglobin thereby alter its oxygen affinity
Lorestan University Biochemistry
•Occasionally 2,3BPG leaves the enzyme •The concentration of 2,3 BPG in erythrocytes is much higher than the amounts need as PGM primer • Erythrocytes synthesize and degrade 2,3 BPG by a detour from the glycolytic pathway
The rate of glycolysis affects the oxygen affinity of hemoglobin through the mediation of 2,3 BPG
Lorestan University Biochemistry
•The next stage is water removal from the 2,PG
Lorestan University Biochemistry
•The next stage is water removal from the 2,PG • enolphosphate compound is a high-energy one; ΔG0´= - 62.2 kJ mol-1
Lorestan University Biochemistry
Lorestan University Biochemistry
Lorestan University Biochemistry
• Reoxidation of cytoplasmic NADH from glycolysis by electron shuttle systems •The NADH generated in glycolysis is reoxidised • Transferring its electrons into mitochondria • NADH itself cannot enter into mitochondria • Inner membrane of mitochondria lacks an NADH transport protein • Two systems for transferring the electrons into mitochondria
Lorestan University Biochemistry
• Glycerol phosphate shuttle • Insect flight muscle • 3-phosphoglycerol dehydrogenase, catalyzes the oxidation of cytosolic NADH • G3P can reach the inner mitochondrial membrane • Different type of G3PDH, built in the inner membrane –FAD as its prosthetic group
Lorestan University Biochemistry
•Malate-Aspartate shuttle • Transfers the electron from cytosolic NADH to mitochondrial NAD+ • Intermediate reduction and subsequent regeneration of oxaloacetate
Lorestan University Biochemistry
•Malate-Aspartate shuttle • Phase A (transport of electrons into the matrix) • Reoxidising the NADH • Transport of malate into mitochondria in exchange for α- ketoglutarate • Reoxidising the malate in mitochondria Lorestan University Biochemistry
•Phase B (regenration of cytosolic oxaloacetate) • Mitochondrial conversion of oxaloacetate to aspartate (concomitant conversion of glutamate to α- ketoglutarate • Transporting the aspartate to the cytosol (in exchange for glutamate)
Lorestan University Biochemistry
•Phase B (regenration of cytosolic oxaloacetate) •Cytosolic conversion of Aspartate to oxaloacetate (in conjugation with conversion of α- ketoglutarate to glutamate)
Lorestan University Biochemistry
• Formation of Acetyl-CoA •The products of gycolysis • NADH, Pyruvate • Pyruvate is transported into mitochondrial matrix • In exchange for OH-, antiport system • Conversion of pyruvate to Acetyl-CoA occurs in five reaction
Lorestan University Biochemistry
• Formation of Acetyl-CoA
• Pyruvate dehydrogenase, the responsible enzyme • Consist of three different enzymes
Lorestan University Biochemistry
Lorestan University Biochemistry
• Formation of Acetyl-CoA • The first step is decarboxylation of pyruvate by pyruvate dehydrogenase a TPP Biochemistry.ppt#151. Slide 151requiring enzyme
• Production of CO2 and a hyroxyethyl (CH3CHOH- ) group •Biochemistry.ppt#157. Slide 157
Lorestan University Biochemistry
Biochemistry.ppt#150. Slide 150
Lorestan University Biochemistry
• Formation of Acetyl-CoA • The second step • Transferring the hydroxyethyl group to the dihydrolipoyl transacetylase Biochemistry.ppt#153. Slide 153 •Biochemistry.ppt#157. Slide 157
Lorestan University Biochemistry
Lorestan University Biochemistry.ppt#152. Slide 152 Biochemistry
• Formation of Acetyl-CoA • The third step
• E2 then catalyze the transfer of acetyl group to CoA, yielding acetyl-CoA and dihydrolipoamideBiochemistry.ppt#153. Slide 153 • Biochemistry.ppt#157. Slide 157
Lorestan University Biochemistry
• Formation of Acetyl-CoA • The forth step
• Dihydrolipoyl dehydrogenase (E3) reoxidizes dihydrolipoamide, compeleting the catalytic cycle of E2
• E3 contains a reactive disulfide group and a tightly bond FAD •Biochemistry.ppt#157. Slide 157
Lorestan University Biochemistry
• Formation of Acetyl-CoA • The fifth step
+ • Reduced E3 is reoxidized by NAD •Biochemistry.ppt#157. Slide 157
Lorestan University Biochemistry
Biochemistry.ppt#150. Slide 150
Lorestan University Biochemistry
• TCA
Biochemistry.ppt#161. Slide 161
Lorestan University Biochemistry
• Topping up the TCA • Starts with oxaloacetate condensing with acetyl-CoA • Ends with oxaloacetate • Oxaloacetate is not used up • Note: • The cycle does not exist in isolation form the rest of metabolism • Some the TCA’s acids are drawn off for other purposes
Lorestan University Biochemistry
• Topping up the TCA • Cycle acids cycle occupy a special place in metabolism • They are not available from the diet in large amounts
• Dietary carbohydrates give rise to large quantities of C3 acid • Pyruvate production
• Cycle acids cycle (C4, C5 and C6) are not available in such quantities
Lorestan University Biochemistry
• Topping up the TCA
• Fats provide large amounts of the C2 (acetyl groups) • Completely destroyed in the cycle • Biochemistry.ppt#158. Slide 158
Lorestan University Biochemistry
• Topping up the TCA • Certain amino acids can provide cycle acids •Biochemistry.ppt#53. Slide 53 • The same taken to synthesize some amino acids
Lorestan University Biochemistry
• Topping up the TCA • To keep energy generating mitochondrial reactions running properly • A topping up method is necessary • There is such a provision in cells • A reaction so-called: Anapleortic or filling up:
• Pyruvate plus CO2 being converted to Oxaloacetate
Lorestan University Biochemistry
• Topping up the TCA •A reaction so-called: Anapleortic or filling up:
• Pyruvate plus CO2 being converted to Oxaloacetate
Pyruvate carboxylase
• Crucial point: C3 acids can be converted to C4 acids
Lorestan University Biochemistry
Pyruvate carboxylase
• Biotin is the cofactor for CO2 activation • Covalently bound to its enzyme • Accept a carboxy group from bicarbonate • Carboxybiotin
ΔG0´ for its cleavage is -19.7 kJ mol-1
Lorestan University Biochemistry
Pyruvate carboxylase
• Have two catalytic sites • One to carboxylate the biotin • One to transfer the carboxy group to pyruvate
Lorestan University Biochemistry
• Topping up the TCA • E. coli does not posses pyruvate carboxylase • They do have TCA • How they can manage without the topping up reaction? •Biochemistry.ppt#56. Slide 56
Lorestan University Biochemistry
• The electron transport chain • Stage 1: Glycolysis • Step 2: TCA • Energy wise we have not achieved much yet: • Only 4 mol ATP per mol glucose • Two from glycolysis • Two from the TCA (via GTP)
• Other products starting with glucose: 10 NADH and 2 FADH2
Lorestan University Biochemistry
• The electron transport chain
• The oxidation of the NADH and FADH2 produce most of the ATP generated by glucose oxidation
• Electron transport chain:
• Its purpose: ATP production from ADP and Pi • Inner membrane of mitochondria
Lorestan University Biochemistry
• The electron transport chain
• Free energy of electron transfer from NADH and FADH2 to O2 • Via protein bound redox centres • Coupled to ATP synthesis
Lorestan University Biochemistry
• We begin with thermodynamics of electron transport • NADH oxidation is highly exergonic reaction • ΔG0´= -218 kJ mol-1 under standard condition • Standard free energy required to synthesize
-1 • 1 mol ATP from ADP + Pi is 30.5 kJ mol
• If oxidation of NADH is coupled to ATP synthesis • Sufficient to drive the formation of several moles of ATP
Lorestan University Biochemistry
•If oxidation of NADH is coupled to ATP synthesis • This coupling achieved by an electron transport chain • Electrons are passed to oxygen • Through three protein complexes instead of directly to
O2 • Having progressively greater electron affinity • Allows the large overall free energy change: • To be broken in three smaller packets • Each of which is coupled with ATP synthesis Oxidative Phosphorylation
Lorestan University Biochemistry
• Oxidative Phosphorylation • Theoretically oxidation of 1 mol NADH should result 3 mol ATP • Thermodynamic efficiency of oxidative phosphorylation under standard conditions (3×30.5/218) × 100 = 42 % • Under physiological condition in active mitochondria • Is thought to be ~70%
Lorestan University Biochemistry
• Sequence of Electron Transport • Necessary energy to generate ATP
• Is extracted from the oxidation of NADH and FADH2: • By the electron transport chain:
• A series of four protein complexes • Electrons pass from lower to higher standard reduction potentials
Lorestan University Biochemistry
Peripheral membrane protein
Lorestan University Biochemistry
Lorestan University Biochemistry
Lorestan University Biochemistry
• The changes in standard reduction potential of an electron pairs as it traverse Complexes Ι, ΙΙΙ,ΙV at each stage • Sufficient energy to power the synthesis an ATP molecule
Lorestan University Biochemistry
• Our understanding of the sequence event in electron transport • Using the specific inhibitors
• The rate at which O2 is consumed by a suspension of mitochondria • A sensitive measure of the functioning of the electron transport chain • Conveniently measurable in an oxygen electrode
Lorestan University Biochemistry
•Conveniently measurable in an oxygen electrode
Lorestan University Biochemistry
•Conveniently measurable in an oxygen electrode • Oxygen disappearance • Compounds that inhibits electron transfer (as judged by their effects on oxygen disappearance) • Invaluable experimental probes in tracing the path of electrons • Invaluable experimental probes in tracing the points of entry of electrons from various substrates
Lorestan University Biochemistry
• Among the most useful such substances • Rotenone ( a plant toxin used by Amazonian Indians) • Amytal (a barbiturate) • Antimycin A (an antibiotic) • Cyanide
Lorestan University Biochemistry
Lorestan University Biochemistry
• Experiment
• A buffered solution containing excess ADP and Pi • Equilibrated in the reaction vessel of an oxygen electrode • Reagents are then injected into the chamber
• Then O2 consumption recorded
Lorestan University Biochemistry
Lorestan University Biochemistry
Lorestan University Biochemistry
• Three jumps between
• NADH, CoQ, cytochrome c, and
O2 are each of sufficient magnitude to drive ATP
synthesis
Lorestan University Biochemistry
• Three jumps in reduction potential between
• NADH, CoQ, cytochrome c, and O2 are each of sufficient magnitude to drive ATP synthesis
• The redox potential jumps corresponds to the points of inhibition of rotenone, Antimycin A, and CN-
Lorestan University Biochemistry
• Phosphorylation and oxidation are rigidly coupled
• The foregoing thermodynamic studies suggest that:
• Oxidation of NADH, FADH2 and ascorbate by O2 • Associated with the syntesis of three, two and one mol ATP • This stoichiometry called the P/O ratio
Lorestan University Biochemistry
• Phosphorylation and oxidation are rigidly coupled
• A suspension of mitochondria containing excess of Pi but no ADP is incubated in an oxygen electrode • Nothing happens since no ADP is present • Conclusion: Oxidation and phosphorylation are closely coupled in well functioning mitochondria
Lorestan University Biochemistry
• Phosphorylation and oxidation are rigidly coupled
• Conclusion: Oxidation and phosphorylation are closely coupled in well functioning mitochondria
Lorestan University Biochemistry
Lorestan University Biochemistry
• Components of the electron transport chain
Lorestan University Biochemistry
• Components of the electron transport chain • Many of the proteins embedded in the inner mitochondrial membrane • Are organised into the four respiratory complexes • Each complex consists of several protein components • Are associated with a variety of redox-active prosthetic group (successively increasing reduction potential)
Lorestan University Biochemistry
• Components of the electron transport chain • The complexes are all laterally mobile within the inner membrane of mitochondria
Lorestan University Biochemistry
• Complex Ι (NADH- Coenzyme Q reductase) • Passes electrons from NADH to CoQ • Probably the largest protein component of the inner membrane • One molecule of FMN ( a redox-active prosthetic group) • Six to seven Iron-Sulfur clusters ( participate in the electron- transport process)
Lorestan University Biochemistry
• Iron-Sulfur clusters are redox active • Prosthetic groups of iron-sulfur proteins ( nonheme iron proteins) • Three types are known • Two most common types • [2Fe-2S], [4Fe-4S] • Both are coordinated to four protein Cys sulfhydryl groups
Lorestan University Biochemistry
Fe atoms in all three types are each coordinated by four S atoms
Lorestan University Biochemistry
• The [Fe-S] cluster has been found only in bacteria
• The oxidised and reduced state in all types
• Differ only by one formal charge regardless of their number of Fe atoms • In Ferredoxin: • Contains one Fe (ΙΙ) and two Fe(ΙΙΙ) in its oxidised form • Contains two Fe (ΙΙ) and two Fe(ΙΙΙ) in its reduced form
Lorestan University Biochemistry
• The Coenzyems of complex Ι • FMN and CoQ each can adopt three oxidation states
Hydrophobic tail
Lorestan University Biochemistry
• NADH can only participate in a two-electron transfer
• Both FMN and CoQ are capable of accepting or donating • Either one or two electrons • Because their semiquinone forms are stable • Cytochrome c to which CoQ passes its electrons • Only capable of one-electron reductions • FMN and CoQ provide an electron conduit between
• The two-electron donor NADH and the one-electron acceptors, cytochromes
Lorestan University Biochemistry
• CoQ’s hydrophobic tail makes it soluble in the inner membrane
• In mammals of 10 C5 isoprenoid units (Q10) • In other organisms, CoQ may have only 6 (Q6) or 8 (Q8) isoprenoid units
Lorestan University Biochemistry
• Complex ΙΙ (Succinate-Coenzyme Q reductase)
• Succinate dehydrogenase • Three other small hydrophobic subunits • Passes electrons from succinate to CoQ
• How?
• With the participation of :
• A covalently bound FAD • One [4Fe-4S] cluster
• Two [2Fe-2S] clusters
• One cytochrome b560
Lorestan University Biochemistry
• Complex ΙΙ (Succinate-Coenzyme Q reductase)
• One iron-sulfur has a standard reduction potential that is too
low to accept electrons from succinate
Lorestan University Biochemistry
• Complex ΙΙ (Succinate-Coenzyme Q reductase)
• Its standard redox potential for electron transport from succinate to CoQ • Is insufficient to provide the free energy for ATP synthesis
• It allows high- potential electrons to enter the electron
transport chain
Lorestan University Biochemistry
• Complex ΙΙΙ (Coenzyme Q- Cytochrome c reductase)
• Passes electrons from reduced CoQ to cytochrome c
• Two b-cytochromes, one cytochrome c1, and one [2Fe-2S] cluster • The heme groups of reduced [Fe (II)] cytochromes have
visible absorption spectra consisting of three peaks
Lorestan University Biochemistry
Lorestan University Biochemistry
• Complex ΙΙΙ (Coenzyme Q- Cytochrome c reductase)
• Within each group of cytochromes • May be characterised by slightly different α peal wavelenghts • Example: Complex ΙΙΙ has • Two b-type cytochrome
• Absorbing maximally at 562 nm (b562) or bH
• Absorbing maximally at 566 nm (b562) or bL
Lorestan University Biochemistry
• Each groups of cytochromes contains a differently subsituted porphyrin ring
• Coordinated with the redox-active iron atom
Lorestan University Biochemistry
• b – types cytochrome contain protoporphyrin ΙX • Also occurs in hemoglobin
Vinyl group
Lorestan University Biochemistry
•Differs from protoporphyrin ΙX • Vinyl groups have added Cys sulfydryl across thei double bonds • To form thioester linkages to the protein
Lorestan University Biochemistry
•Differs from protoporphyrin ΙX • Consists of a long hydrophobic tail of isoprene units attache to porphyrin • A formyl group in place of methyl substitute
Lorestan University Biochemistry
• Complex ΙΙΙ (Coenzyme Q- Cytochrome c reductase)
• Both cytochrome c and nonheme iron-sulfur protein (ISP) • Located on the membrane’s outer surface • Cytochrome b is a transmembrane protein • Contains both b-type cytochrome hemes
• bH and bL, associate with a single polypetide chain
• The heme group closer to the cytoplasmic side, bL
• The heme group closer to the matrix side, bH
Lorestan University Biochemistry
• Complex ΙΙΙ (Coenzyme Q- Cytochrome c reductase)
• Cytochrome c is a peripheral protein
Lorestan University Biochemistry
• Complex ΙV (Cytochrome c oxidase)
• Catalyse the one-electron oxidation of:
• Four consecutive reduced cytochrome c
• Four-electron reduction of one O2 molecule
Lorestan University Biochemistry
• Complex ΙV (Cytochrome c oxidase)
• Composed of 6 to 13 subunits • Suunits I and II contain all four of its redox-active centres: 2+ • Two a-type hemes (a and a3) that alternates between their Fe and Fe3+ oxidation states • Two Cu atoms, alternate between +1 and +2 oxidation states
• Heme a and the Cu atoms designated CuA
• Heme a3 and the Cu atoms designated CuB
Lorestan University Biochemistry
• CuA is liganded to subunit I
• Through two Cys and two His residues • Is part of cytochrome-c binding site
• CuB and heme a3 are bridged by an S atom
• Form a binuclear complex, comprises
O2 binding site
Lorestan University Biochemistry
• Complex ΙV (Cytochrome c oxidase)
• The reduction of O2 to 2 H2O by cytochrome c oxidase takes palce:
• On the cytochrome a3-CuB binuclear complex
• The reaction consists of four consecutive one-electron transfers
• From the CuA and cytochrome a sites
Lorestan University Biochemistry
• 1. & 2. The binuclear
Fe(III)a3 – Cu(II)B is reduced by two one-electron transfers from Cyt c • By cytochrome a and
CuA
• Formation of Fe(II)a3 –
Cu(I)B
Lorestan University Biochemistry
• 3. O2 binds to this reduced binuclear complex
• To bridge its Fe(II)a3 and Cu(I)B atoms
Lorestan University Biochemistry
• 4. Electron redistribution rapidly yields • Peroxy adduct Fe (III) – O-- O- - Cu(II)
• 5. A further one-electron transfer together with acquisition of a proton converts the adduct to Fe (III) – O-- OH - Cu(II)
Lorestan University Biochemistry
• 6. The acquisition of the second proton and an electron rearrangement results in 2– Fe (IV) = O H2O - Cu(II) (Fe (IV) is said to have ferryl oxidation state)
• 7. The forth one electron transfer together with proton rearrangement yields - – - Fe (III) - OH Cu(II)- H2O -OH
Lorestan University Biochemistry
• 8. Finally, the acquisition of two more protons yields 2 H2O together with Fe(III)a3 – Cu(II)B complex, completing the cycle
Lorestan University Biochemistry
• Oxidative phosphorylation
• The endergonic synthesis of ATP form ADP and Pi
• Catalyzed by proton translocating ATP synthase (Complex V)
• Complex V is physically distinct from the proteins mediating electron transport (complexes I-IV) • The free energy released by electron transport must be conserved • So that ATP synthase can utilise it • Such energy conservation is referred to as • Energy coupling or Energy transduction
Lorestan University Biochemistry
• Energy coupling hypothesis
• The chemical coupling hypothesis • Proposed by Edward Slater (1953) • Electron transport yields reactive intermediates • Their subsequent breakdown drive oxidative phosphorylation • Such a mechanism is responsible for ATP synthesis in glycolysis • Exergonic oxidation of glyceraldehyde 3-phosphate by NAD+ yields 1,3 DPG
Lorestan University Biochemistry
• Energy coupling hypothesis •Exergonic oxidation of glyceraldehyde 3-phosphate by NAD+ yields 1,3 DPG • 1,3 DPG is a reactive (high energy) acyl phosphate • Its phosphoryl group is transferred to ADP to form ATP
• It is abounded • No appropriate reactive intermediates have been found
Lorestan University Biochemistry
• The conformational-coupling hypothesis •Formulated by Paul Boyer (1964) • Electron transport causes the proteins of the inner membrane • To assume “activated” or “energized” states • These proteins are somehow associated with ATP synthase • Such that their relaxation back to the deactivated conformation state • Drives ATP synthesis
• Has found little experimental support
Lorestan University Biochemistry
• The chemiostatic hypothesis • Proposed by Peter Mitchell (1961) • The model most consistent with the experimental evidence • The free energy of electron transport is conserved, How? • By pumping H+ from the mitochondrial matrix to intermembrane space • Creates an electrochemical H+ gradients across the inner mitochondrail membrane • Electrochemical gradient is harnessed to synthesize of ATP
Lorestan University Biochemistry
•Electrochemical gradient is harnessed to synthesize of ATP
Lorestan University Biochemistry
• The chemiostatic hypothesis • Applying this concept, some consideration should be taken into account for ATP generation: • Electron transport must create a gradient of some sort • The gradient must be allowed to flow back through a device • Uses the energy of the gradient to synthesize ATP • Existence of intact vesicle across whose membrane a gradient can be established
Lorestan University Biochemistry
• The chemiostatic hypothesis • Several key observation can be explained by this theory • Oxidative phosphorylation requires an intact inner mitochondrial membrane • The inner membrane is impermeable to ions such as H+ , OH-, K+ and Cl- • Electron transport results in the transport of H+ out of intact mitochondria • Compounds that increase the permeability of the inner membrane mitochondria to proton • Dissipate the electrochemical gradient
Lorestan University Biochemistry
• The chemiostatic hypothesis • Several key observation can be explained by this theory •Compounds that increase the permeability of the inner membrane mitochondria to proton • Dissipate the electrochemical gradient • Allow the electron transport to continue • But inhibit the ATP synthesis
Lorestan University Biochemistry
• Proton gradient generation • Electron transport • Causes complexes I, III and IV to transport protons across the inner membrane • From the matrix, region of low [H+] and negative electrical charge • To the intermembrane space, a region of high [H+] and positive electrical charge • The free energy sequestered is called proton motive force
Lorestan University Biochemistry
• Proton pumping is an endergonic process • The free energy of transporting a proton out of the mitochondria • Against electrochemical gradient is expressed by
• Z is the charge on the proton (including sign) • ΔΨ is the membrane potential • When an ion is transported from negative to positive, ΔΨ is positive
Lorestan University Biochemistry
• Proton pumping is an endergonic process •
• The pH out is less than pH in • The export of protons from the mitochondrial matrix is an endergonic process • Liver mitochondria • membrane potential across the mitochondria, 0.168 (corresponds to an ~ 210000 V.cm-1 across its ~80Å thickness) • The pH of its matrix is 0.75 units higher than that of its intermembrane space • ΔG for proton to transport out, 21.5 kJ mol-1
Lorestan University Biochemistry
• Mechanisms proposed for proton transport • Three of the four electron-transport complexes are involved • Complexes, I, III and IV • Mechanisms that would couple the free energy of electron- transport with active transport of protons • Redox loop mechanism • Proton pump mechanisms
• The redox loop mechanism
Lorestan University Biochemistry
• The redox loop mechanism • Proposed by Mitchell • Redox centres of the respiratory chain (FMN, CoQ, cytochromes, and iron-sulfur clustres) • Be so arranged that • Reduction would involve a redox centre: • Simultaneously accepting e- and H+ from the matrix side • Reoxidation of this redox centre by the next centre in the chain • Releasing the H+ on the cytosolic side together with the transfer of electrons back to the matrix side
Lorestan University Biochemistry
•Releasing the H+ on the cytosolic side together with the transfer of electrons back to the matrix side
Lorestan University Biochemistry
• The electron flow from one carrier to the next one • Yields net translocation of H+ • Creation of an electrochemical gradient (ΔΨ and ΔpH)
• The redox loop mechanism requires • First redox carrier contains • More hydrogen atoms in its reduced state than in its oxidised state • Second redox carrier • Have no difference in its hydrogen atom content between its reduced and oxidised states
Lorestan University Biochemistry
• Are these requirements met in the electron-transport chain? • FMN and CoQ contain more hydrogen atoms in their reduced state than in their oxidised state • Proton carriers as well as electron carriers • If these were spatially with pure electron carriers (cytochromes, Iron-sulfur clusters) • The mechanism could well be accommodated
Lorestan University Biochemistry
•The mechanism could well be accommodated
Lorestan University Biochemistry
• The main difficulty with the redox loop mechanism + - - • The deficiency of ( H + e ) that can alternate with pure e carriers • 15 pure electron carriers • Up to 8 iron-sulfur proteins • 5 cytochromes • 2 Cu atoms • Only two H+ + e- are known
Lorestan University Biochemistry
• The main difficulty with the redox loop mechanism
• There are 3 complexes with standard-reductions potential • Large enough to provide free energy for ATP synthesis • That suggest: The need for at least three proton- transport sites
Lorestan University Biochemistry
•That suggest: The need for at least three proton- transport sites • The problem is emphasized by showing X as an unknown ( H+ + e-) carrier
Lorestan University Biochemistry
Lorestan University Biochemistry
• Thus half the electrons liberated by oxidation of CoQH2 to
CoQ are used to reduced Q to QH2
Lorestan University Biochemistry
• The result of Q cycle in complexIII
• Transport of two protons for each electro transferred
• from complex I via CoQH2 to cytochrome c1
+ - • The CoQ cannot operate in complex IV (contains no H + e carriers)
• But pumps proton from matrix to cytosolic side
Lorestan University Biochemistry
• The proton pump mechanism • Does not require that the redox centres themselves be the hydrogen carriers
• According to this model • Transport of electrons results in conformational changes to the complex • The translocation of protons occurs as a results of the influence of these conformational changes: • On the pK’s amino acid side chains and their alternate exposure to the internal and external side of the membrane
Lorestan University Biochemistry
Lorestan University Biochemistry
• One documented proton pump is the • Bacteriorhodopsin Halobacter halobium • Has seven membrane-spanning helical segments • Forming a polar channel • The free energy required for pumping protons • Obtained through the absorption of light by its retinaldehyde-Schiff base prosthetic group
Lorestan University Biochemistry
Lorestan University Biochemistry
• The same mechanisms is thought to operate in cytochrome c oxidase • Oxidation- reduction causing the pK-altering conformational change
Lorestan University Biochemistry
• ATP synthesis by ATP synthase is driven by • Proton gradient
• ATP synthase is the name of the structure with • One part visible as a knob • Projecting into the matrix on the inside surface of inner
membrane (F1 unit) • The other part anchored in the membrane itself
Lorestan University Biochemistry
Lorestan University Biochemistry
• The reaction catalysed by the ATP synthase
• The standard free energy is +29.3 kJ • Cannot proceed without a large energy input • Supplied by the proton gradient • Established across the membrane by electron transport
Lorestan University Biochemistry
Lorestan University Biochemistry
•ATP synthase is found in aerobic organism • Wherever the energy from electron transport has to be trapped as • ATP
Lorestan University Biochemistry
Lorestan University Biochemistry
•ATP synthase
• The F1 unit, its role in the conversion of ADP +Pi to ATP
• F1 unit • Is a ring formed by six protein subunits • Arrange in a barrel-like structure • The knob consists of a hexamer • Three α protein subunits • Three β protein subunits
Lorestan University Biochemistry
• β subunits • Has a catalytic site which synthesizes ATP from ADP and
Pi • The narrow cavity of the barrel • Is occupied by an elongated asymmetric shaft • The γ subunit • Another subunit called ε
• Forms part of the stalk structure
Lorestan University Biochemistry
• Activities of the enzyme catalytic centres on the F1 unit • The sequence of events in the synthesis of a an ATP on a single β subunit • The site is open and nothing is bound to it (O state) • Conformational change in the protein • Converts the site to a low - affinity state
• ADP and Pi bind to it loosely (L state) • But there is no catalytic activity
Lorestan University Biochemistry
• A further conformational change • Produces a tight binding state
• The ADP and Pi become tightly bound • Catalytically active and ATP is formed
Lorestan University Biochemistry
• Energy is needed to release ATP • Supplied by conformational changes which the enzymes undergoes during the catalytic cycle • Caused by the rotating γ subunit
Lorestan University Biochemistry
• Structure of F0 subunit • Built in the inner membrane • Is the motor which is driven by a flow of proton • From the outside to the inside of the mitochondria
• It rotates the γ subunit inside the F1 subunit • Consisting a ring of c subunits • 10 to 14
Lorestan University Biochemistry
• Mechanism by which proton flow causes rotation of F0
Lorestan University Biochemistry
Lorestan University Biochemistry
• It is essential to note that c ring is surrounded by the hydrophobic lipid bilayer • Except for the two c subunit interfacing with the a protein • 10 subunits are in the hydrophobic environment • Energetically requires the central aspartyl residue of each of these to be • Protonated (uncharged) state (COOH) rather than charged state (COO-)
Lorestan University