Oxidative Phosphorylation and Mitochondria Transport Systems Mitochondria = power house of the cellglyco. TCA NADH, FADH2 (energy rich mols)f.a.oxi. each has a pair of e- (having a transfer pot.) 2 e- 02 Energy released! (used for ATP)Oxidative Phoshorylation: the process in which ATP isformed as electrons are transferred from NADH or FADH2to O2 by a series of electron carriers
Some Features…1. Oxidative phosphorylation is carried out by respiratory assemblies that are located in the inner membrane. – TCA is in the matrix2. The oxidation of NADH 2.5 ATP3. FADH2 1.5 ATP – Oxidation and phosphorylation are COUPLED4. Respiratory assemblies contain numerous electron carriers – Such as cytochromes5. When electrons are transferred H+ are pumped out6. ATP is formed when H+ flow back to the mitochondria
Some Features Continued… Thus oxidation and phosphorylation are coupled by a proton gradient across the inner mitochondria membrane – So, we produce ATP through this – Glycolysis and TCA cycle can continue only if NADH and FADH2 are somehow reoxidized to NAD+ and FAD
Release of Free Energy During Electron Transport1. Electrons transferred electron donor (reductant) electron acceptor (oxidant) They can be transferred – H- – H+ – Pure electrons2. When a compound loses its electrons becomes oxidant cyt b (Fe ++) + cyt c1 (Fe +++) cyt b (Fe +++) + cyt c1 (Fe ++) red. X oxi. Y oxi. X’ red. Y’ Red. X and Oxi. X’ Redox Red. Y’ and Oxi. Y Pairs
Release of Free Energy Continued…3. PAIRS differ in their tendency to lose electrons – It is a characteristic of a pair – Can be quantitatively specified by a constant… E0 (volts) – E0: standard reduction potential – The more negative E0, the higher the tendency of the reductant to lose electrons – The more positive E0, the higher the tendency of the oxidant to accept electrons – Electron transfer: more –E0 ---------- more +E04. Free energy decreases as electrons are transferred Go = -nF E0 where “n” is the number of electrons transferred, and F is Faraday’s constant (23, 062) E0 = E0 (electron accepting pair) – E0 (electron donating pair)
What Are the Electron Carriers in mt? Most of the electron carriers in mitochondria are integral proteins There are four types of electron transfers 1. Direct transfer of electrons Fe+3 Fe+2 2. As a hydrogen atom H+ + electron 3. As a hydride ion :H- (has 2 electrons) 4. Direct combination of an organic reductant with O2
Flow of electrons and protons thru the respiratory chain
How Is This Order Found?1. NADH, UQ, cytb, cytc1, c, a, and a3 is the order – Their standard reduction potentials have been determined experimentally! – The order increased E0 because electrons tend to flow from more negative E0 to more positive E02. Isolated mitochondria are incubated with a source of electrons but without O2 – a, a3 is oxidized first – c, c1, b are second, third, and fourth respectively – When the entire chain of carriers is reduced experimentally by providing an electron source but no O2 (electron acceptor) then O2 suddenly introduced into the system – The rate at which each electron carrier becomes oxidized shows the order in which the carriers function – The carrier nearest O2 is oxidized first, then second, third, etc.
Action of Dehydrogenases Most of the electrons come from Electron acceptors NAD or FMN, FAD Reduced subs + NAD+ ox. sub + NADH + H+ Reduced subs + NADP+ ox. Sub + NADPH + H+ In addition to FAD and NAD, there are three other types of electron carrying groups – Ubiquinone – Iron containing proteins (cytochromes, Fe-S proteins) Ubiquinone = CoQ or = UQ – When it accepts 1 electron UQH (semiquinone) – When it accepts 2 electrons UQH2 (ubiquinal)
Oxidation states of flavins.• The reduction of flavin mononucleotide (FMN) to FMNH2 proceeds through a semiquinone intermediate.
Complex I NADH dehydrogenase (NADH Q reductase) Huge protein – 25 pp FMN, Fe-S I electron UQ
Complex II Succinate Q Recuctase (Succinate dhydrogenase) – Is the only membrane bound enzyme in the TCA cycle – Contains FAD, Fe-S II electrons UQ Cytochrome: an electron transferring protein that contains a heme prosthetic group!
Complex III Cyt reductase (UQ-cyt c oxido reductase or cyt bc1 complex) – Contains cyt b, c1, Fe-S proteins and at least six other protein subunits UQ is 2e- carrier, cyts are 1e- carriers – This switch is done in a series of reactions (called Q cycle) Electron transfer in III seems to be complicated but it’s not Net reaction: – UQH2 UQ and cyt c is reduced – H+ is pumped out also
Complex IV Cyto oxidase – Contains a, a3, and CuA, CuB The detail of this electron transfer in Complex IV is not known It also functions as a proton pump
ATP Production in Mitochondria
Mitchel’s Theory The electrochemical potential difference resulting from the asymmetric distribution of the H+ is used to drive the mech. responsible for the formation of ATP
Chemiosmotic Theory Continued… G = RT ln(C2/C1) + ZF [ + ] [ + ]When H+ is pumped against electrochemical gradient G=+ When protons flow back inside, this G becomes available to do the work!!
Oxidation and ATP synthesisare coupled
Uncoupled Mitochondria in Brown Fat Produces Heat This is done by DNP or other uncouplers They carry protons across the inner mitochondria membrane In the presence of DNP, electron transport is normal but ATP is not formed Proton-motive force is gone or disrupted Uncoupling is also seen in brown adipose tissue It is useful to maintain BT in hibernating animals, newborns, and mammals adapted to the cold It has lots of mitochondria IMM thermogenin (uncoupling protein) Thermogenin generates heat by short-circuiting the mitochondrial proton battery
Shuttle Systems Required for cytosolic NADH oxidation NADH dehydrogenase IMM can accept electrons only from NADH in the matrix We also make cytosolic NADH by glycolysis They also have to be reoxidized to NAD+ IMM is not permeable to cytosolic NADH – We therefore need shuttle systems Electrons are transferred from NADH to Complex III (not I), providing only enough energy to make 2 ATP (G-3-P shuttle) It is active in muscle (insect flight) and brain Net reaction: – NADH + H+ + E- FAD NAD+ + E-FADH2 (cytosolic) (mitochondrial) (cyto) (mito) So, 2ATP is formed UQ
Malate-aspartate Shuttle Heart Liver Cytoplasmic NADH is brought to mitochondria by this shuttle This shuttle works only if NADH/NAD+ increase in the cytosol (then mitochondria) No energy consumed No ATP lost
Regulation of ATP ProducingPathways Coordinately regulated – Glycolysis – TCA – FA oxidation – a.a. oxidation – Oxidative phosphorylation Interlocking regulatory mech. ATP, ADP controls all of them Acetyl CoA and and citrate
Regulation of Oxidative Phosphorylation Intracellular [ADP] If no ADP no ATP – The dependence of the rate of O2 consumption on the [ADP] (Pi acceptor) is called “acceptor control” acceptor control ratio = ADP-induced O2 consumption O2 consumption without ADP Mass action ratio: ATP is high normally [ADP][Pi] So, system is fully phosphorylated.ATP used, ratio decreases, rate of oxidative phosphorylationincreases.
Tumor Cells Regulation is gone in catabolic processes Glycolysis is faster than TCA They use more Glc, but cannot oxidize pyruvate Pyruvate lactate (PH decreases in tm.)
Mutations in Mitochondrial Genes Mutations in mitochondrial genes cause human disease. DNA has 37 genes (16, 569 bp), 13 of them encode respiratory chain proteins. LHON- Leber’s Hereditary Opti-neuropathy – CNS problems – Loss of vision – Inherited from women. – A single base change ND4 Arg His (Complex 1) – Result: defective electron transfer from NADH to UQ. Succinate UQ okay, but NADH UQ not.
3 Stages ofCatabolism
Summary Electron flow results in pumping out H+ and the generation of membrane potential! ATP is made when protons flow back to the matrix! F0F1 complex Proton motive force, PH gradient, membrane potentialThe flow of two electrons through each of threeproton-pumping complexes generates a gradientsufficient to synthesize one mole of ATP!
The proton gradient is an interconvertible form of free energy Proton gradients are a central interconvertible currency of free energy in biological systems. • Active transport of Ca • Rotation of bacterial flagella • Transfer of e from NADP+ to NADPH • Generate heat in hybernation