Mitochondria.Mechanism of oxidative Mechanism of oxidative phosphorylationp p y

The mitochondrion has been termedhas been termedthe "powerhouse" of the cell.

Mitochondria are oval-shaped organelles. They have 2 membrane systems:outer membrane and inner membrane. The wrinkles, or folds are organized into cristae.Th b t tThe membranes create two compartments:The intermembrane space, the region between the inner andregion between the inner and outer membranes. The matrix.

Structure of outer membrane:50% lipids,50% proteins.

The outer membrane is a relatively simple h h li id bil t i i t iphospholipid bilayer, containing protein

structures called porins, which render it permeable to molecules of about 10 kilodaltonsor less (the size of the smallest proteins). Ions,or less (the size of the smallest proteins). Ions, nutrient molecules, ATP, ADP, etc. can pass easy through the outer membrane.

Structure of inner membrane:20% lipids,80% proteins.The inner membrane is freely permeable only to O2 and CO2 .

Its structure is highly complex, including all of the complexes of the electron transport system, the ATP synthetase complex, and transport proteins.transport system, the ATP synthetase complex, and transport proteins.

The matrix contains the enzymes that are responsible for the citric acid cycle reactions, PDH, the enzymes of fatty acid oxidation etc.

Mitochondrial electron transport chain

The main function of respiratory chain – to liberate gradualy energy from reduced coenzymes (NADH ir FADH ) which are substrates of respiratory chainFADH2), which are substrates of respiratory chain.

Mitochondrial electron transport chain

Organization of the electron transport Organization of the electron transport chain

Four complexes:I complex – NADH dehydrogenase, p y g ,II complex - succinate dehydrogenase, III complex cytochromes bc1III complex - cytochromes bc1, IV complex - cytochromoxidase.

They are hydrophobic integral inner membrane proteins.

Electrons are carried by 2 smaller mobile carriers – coenzyme Q and y Qcytochrome c.

III Complex IIIIII IV

FADH2I

Complex I, (NADH:CoQ oxidoreductase or NADH dehydrogenase):dehydrogenase):Protein part - 46 polypeptides.Cofactors - FMN and 22-24 iron-sulfur (Fe-S) centersCofactors - FMN and 22-24 iron-sulfur (Fe-S) centers. Complex I is responsible for transferring electrons from NADH to CoQ.NADH to CoQ.

IIIIIIII IV

FADH2I

Complex II (succinate dehydrogenase or succinate:CoQoxidoreductase):Protein part - 4 polypeptides,cofactors – FAD, 7-8 Fe-S, cytochrome b560. Complex II transfers electrons from succinate to CoQ.

CoQ (ubiquinone) transfers electrons between compex I or II and cytochrome b.CoQ is restricted to the membrane phase. CoQ can

d ith 1 2 l t ti l di tundergo either 1- or 2-electron reactions leading to formation of the reduced quinol, the oxidized quinone, and the semiquinone intermediateand the semiquinone intermediate.

IIIII IV

IFADH2

I

Complex III,The main components of complex III: the heme proteins known as cytochromes b and c1 and a non heme iron protein known ascytochromes b and c1 and a non-heme-iron protein, known as the Rieske iron sulfur protein. The heme iron of all cytochromes participates in the cyclic redoxreactions of electron transport, alternating between the oxidized (Fe+3) and reduced (Fe+2) forms.

IIIII IVII IV

FADH2I

Th l i f C l III tThe electron carrier from Complex III to Complex IV is the smallest of the cytochromes,

t hcytochrome c.

IIIII IV

FADH2I

Complex IV (cytochrome oxidase) contains:the hemoproteins known as cytochrome a andp ycytochrome a3;the copper-containing proteins in which the copper gundergoes a transition from Cu+ to Cu2+ during the transfer of electrons through the complex to molecular oxygen.

Electron transport and H+ release across the Electron transport and H+ release across the inner membrane of mitochondria

Cyt cCyt.c

Consequences of electron transfer across the qinner membrane of mitochondria

The matrix site of the inner membrane of mitochondria gains negative charge because of excess in OH-.g g gThe intermembrane site of the inner membrane of mitochondria gains positive chargeThe difference of both the electric charge and pH isThe difference of both the electric charge and pH is considered as electrochemical gradient of protons or a proton motive force (Δp).

Δp = ΔΨ+ΔpH

Functions of proton motive force Functions of proton motive force (Δp)

Principle of ATP synthesis

ATP synthase. ATP synthase is composed of 3 fragments:fragments: F0, which is localized in the inner membrane;F1, which protrudes from the inside of the inner membrane into the matrix;into the matrix; and oligomycin sensitivity-conferring protein (OSCP),which connects F0 to F1which connects F0 to F1.

Mechanism of ATP synthase Mechanism of ATP synthase functioning

Proton chanel permits protons to return into the t imatrix.

Proton flow results in conformational changes of catalytic subunit.Conformational changes benefit to ADP gphosphorylation.

In order for oxidative phosphorylation to proceed twoIn order for oxidative phosphorylation to proceed, two principal conditions must be met:First the inner mitochondrial membrane must beFirst, the inner mitochondrial membrane must be physically intactSecond, a high concentration of protons must beSecond, a high concentration of protons must be developed on the outside of the inner membrane.

The energy of the gradient is used to drive ATP th i th t t t d b k dsynthesis as the protons are transported back down

their thermodynamic gradient into the mitochondrion.

Mechanism of oxidative phosphorylationThe Chemiosmotic Theory of oxidativeThe Chemiosmotic Theory of oxidative phosphorylation (1961) :Coupling of electron transfer to ATP synthesis is indirect, via a H+

electrochemical gradient:electrochemical gradient: Respiration: Spontaneous electron transfer through complexes I, III, and IV is coupled to non-spontaneous H+ ejection from the mitochondrial matrix. H+

ejection creates a membrane potential (Δϕ, negative in the matrix) and a pH gradient (ΔpH alkaline in the matrix)gradient (ΔpH, alkaline in the matrix). F1Fo ATP Synthase: Non-spontaneous ATP synthesis is coupled to spontaneous H+ transport into the matrix compartment. Respiration drives ATP synthesis indirectly, by creating the pH and electrical gradients that together y y, y g p g gare the driving force for H+ uptake. Return of protons to the matrix via Fo "uses up" the pH and electrical gradients.

Intermembranespace

Matrix

The Adenine Nucleotide Translocase (ADP/ATP carrier) is an antiporter that catalyzes exchange of ADP for ATP across the inner mitochondrial membrane. Phosphate reenters the mitochondrial matrix with H+ by anPhosphate reenters the mitochondrial matrix with H+, by an electroneutral symport mechanism.

Efficiency of Oxidative yPhosphorylation

Th P/O ti i di t h i iThe P/O ratio indicates, how many inorganic phosphate are converted to ATP when 1 atom

f O i dof O2 is consumed. P/O = 3 for NADHP/O = 2 for FADH2

Regulation of Oxidative Phosphorylationg p y

The flow of electrons through the electron transport system is regulated by the magnitude of the PMF. Under resting conditions, with a high cell energy charge, the demand for new synthesis of ATP is limited and, although the PMF is high, flow of protons back into the mitochondria through ATP synthase is minimal. When energy demands are increased, cytosolic ADP rises and is exchanged with intramitochondrial ATP. Increased intramitochondrialconcentrations of ADP cause the PMF to become discharged as protons pour through ATP synthase, regenerating the ATP pool. Thus, while the rate of electron transport is dependent on the PMF, the magnitude of the PMF at any moment simply reflects the energy charge f th llof the cell.

Sites of the ETC Sites of the ETC inhibition

Cyanide,

Antimycine A

Cyanide, CO,AzideNO

RotenoneAmytal

NADH

Uncouplers & IonophoresUncouplers & Ionophores:

ll hi thi l lsmall amphipathic molecules which dissolve in phospholipid bilayers andphospholipid bilayers and increase their ionic permeability.yIonophores transport a variety of ions, uncoupling agents specifically increase the proton permeability and disconnect the electrondisconnect the electron transport chain from the formation of ATP.formation of ATP.

Mechanism of uncoupling in brown adipose tissueMechanism of uncoupling in brown adipose tissue

UCP-1Uncoupling protein

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Mechanisms of transfer of NADH electrons into mitochondria:

1) glycerol phosphate shuttle2) malate-aspartate shuttle2) malate-aspartate shuttle