membrane protein from halobacter can pumps protons when illuminated (expose to light ).

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1 بسم هللا الرحمن الرحيم *bacteriorhodopsin(protein from bacteria): it is a purple membrane protein from halobacter can pumps protons when illuminated (expose to light ). - we took the bacteriorhodopsin and put it in synthetic vesicle and put mitochondrial ATPase(from beef heart mitochondria) in the same vesicle,this vesicle can synthesis ATP without any oxidation. *ATP synthase: large multisubunit enzyme complex when it is isolated from the mitochondria it called originally mitochondria ATPase or Fo F1 ATPase. -because we isolated from mitochondria it has an enzyme responsible for reaction without proton gradient (not depend on it) so it fever ATP hydrolysis rather than ATP synthesis but when it found in proper mitochondria it fever ATP synthesis. -ATP synthase is formed from two subunits which are separated (Fo,F1)..F1:found on mitochondria it can catalyzed hydrolysis (catalytic activity)..f0:found on membrane of mitochondria and it inhibit synthesis because it bind olgiomicine so when you put olgiomicine on intake(proper)mitochondria it inhibit ATP synthesis. -Fo formed from 12c and 1a subunits,12c they form ring and when they come with 1a they(12c+1a)together form proton channel.

2 -F1 head piece are formed from(five type of polypeptide): 1.three α 2. three β 3. 0ne γ 4.one δ 5.one ε

3 _α, β are symmetrical domain alternate but actually are not symmetrical because they attach to γ which is asymmetrical and make them asymmetrical. _α, β make ring similar together before put γ between them, so the places that contact α, β with γ are different to each other. -the Fo and F1 subunits are connected in two ways: 1.central γ ε stalk 2.an exterior column(β) -the proton channel depends on both the α subunit and the c ring. proton channel is not continuous in α there are two half channel. in the center of each channel there are aspartic or glutamic acid(coo -(. There are high concentration of proton because there are a lot of proton pumps so COO will bind with proton and make COOH.COOH is more hydrophobic (non-polar) it can cause rotation (enable to interact with lipid)from here we can expect the c ring is receptor and rotator for proton and it will happen rapidly, after that the proton will leave the c ring and flow to the other half channel to go to matrix side and it make 2.5 ATP release in 365 degree of rotation. - α, β dimers have three different conformations T(tight), L(loose which bind with ADP and Pi), O(open). When c ring rotate the γ will rotate and it change their contact with α, β and change the α, β conformation. When conformation L will change to T and ADP+Pi become ATP and it is difficult to disassociated then T will becom O which is most easy to ATP to disassociated. *oxidation and phosphorylation are tightly coupled by proton motor force.if all ADP are converted to ATP(ATP will stop to produced)the proton flow will stop. Oxidation will stop when the concentration of proton will exceed the energy that produced so the phosphorylation will stop. *the tightly coupled between oxidation and phosphorylation control of oxidative phosphorylation.

4 *when ATP consume stop the oxidation will stop and NADH level will increase so all pathway that produce NADH will stop for example: consuming fuel. Control of oxidative phosphorylation is one way to control the metabolisms as well. *respiratory control: electrons do not flow through electron transport chain to O2 unless ADP will convert to ATP. *oxidative phosphorylation requires: 1.source of electrons (NADH or succinate) 2.ADP 3.Pi 4.O2 If any one of them not found the oxidative phosphorylation does not occur. We isolated mitochondria to measure the O2 consumed it will increase when we add ADP after that ADP will convert to ATP (supply of ADP nearly exhausted )the O2 consumed will decrease. If we add ADP+Pi alone there are not O2 consumed and no ATP synthesis because we need substrate to be oxidized but if we add succinate the rate of O2 consumed will increase

5 and ATP will synthesis(there are coupled of oxidative phosphorylation). After that if we add CN- it block and stop oxidation and ATPase because phosphorylation depend on oxidation as we know. Blocking of oxidation at terminal electrons receptor (cytochrome of oxidize )will stop both oxidation and phosphorylation. *we can measure PO ratio(how much of ATP synthesized by consumed one oxygen atom). Oxygen will be reduced by NADH or succinate produce 2.5 ATP. *NADH: 2.5 ATP *FADH2: 1.5 ATP *SO from one glucose molecule 30 ATP rather than 36 ATP because oxidative phosphorylation coupled by proton gradient not chemically. *if the proton leak back to the matrix by pores in lipid bilayer we make it not by ATPase the oxidation and phosphorylation become uncoupled and the oxidation will continue but the ATPaes will stop. *chemical uncouplers of oxidative phosphorylation:

6 There are lipid soluble (weak acid compound )which can penetrate the lipid bilayer. because they are weak acids they can dissociated to proton and conjugate base EXAPLE: DNP because it has phenol on it, it is lipid soluble and conjugate base is lipid soluble also so can penetrate to intremembrane space and accept proton which is very high concentration in intremembrane space of protons. *and we use DNP to transport proton from intremembrane space to matrix because after conjugated base bind to proton and become DNP it will go to matrix from lipid bilayer. We add succinate alone so there are not oxidative phosphorylation then we add ADP +Pi oxidation and phosphorylation will increase. If we add venturieidin or oligomycin the oxidation and phosphorylation will stop (coupled) but if we add DNP the oxidation will increase and phosphorylation remain stop(uncoupled). *uncoupling proteins(ucp) and thermogenesis:

7 Form channels through the membrane and conduct proton from intermembrane space to matrix. *UCP1:thermogenin in brown and adipose tissue. *UCP2, UCP3,UCP4, UCP5 in other tissue including muscles. *thermogenesis make oxidative phosphorylation uncoupled so there are oxidation without phosphorylation, rather than of phosphorylation there are heat energy released. *brown adipose tissue we find it in new born it is brown because it is rich in mitochondria, it produce heat for new born because she\he cannot make in other way like movement, so after growing the brown adipose tissue will decreased and disappear. *adrenaline (epinephrine) hydrolysis fat to fatty acid which activate UCP1 channel (so it is not always active). *the persons who have a lot of UCP they are lucky because they can eat without making fat from exceed calorie. *most of NADH are produced in matrix of mitochondria, but NADH can be produce in cytosol by glycolic dehydrogenase but this NADH cannot transport to mitochondria matrix because there are not channel for it and its quantity is low. *just 10% from the NADH in the cell are produced in cytosol. The mechanisms: First one

8 The NADH from cytosol will produce 1.5 ATP because it does not pass through complex one but it go to FADH2 (which produce 1.5 ATP Second one:

9 The NADH will give its electrons to malate and malate can inter to matrix to produce 2.5 ATP. *When concentration of NADH high the malate will enter. *This figure named aspartate -malate shuttle.

10 OxPhos dieasis: *oxidative phosphorylation diseases. *mitochondria is like a bacteria it has own DNA (mtdna)encoded 13 subunits of complexes I,III,IV not all proteins of mitochondria encoded by mtdna. *producing of I,III,IV required: 1.mtDNA 2.neuclear DNA * high rate of mutations (10x nuclear DNA ) defect in oxidative phosphorylation (I,III or IV become functionless). *tissue with highest ATP demands are affected most (like: muscles, liver, kidney, brain). *mutation of mitochondrial DNA cannot be repaired. *accumulation of somatic mutations with age(by replications). *maternal inheritance : it pass from mothers because ovum has large number of mitochondria not from fathers because during fertilization they give head of sperm which does not contain mitochondria so all mitochondria in our bodies are from mothers. * examples not to save : 1.leber shereditary optic neuropathy affect CNS 2.myoclonic epilepsy and ragged- red fiber disease (MERRF) affect cardiac and skeletal muscles. Done by: Malik Alkharabsheh *ال تدع الطب يأخذ كل حياتك و وقتك بل اخرج للحياة وأعط لنفسك حقها بالرفاهية وأعط لمن يحبك الحق واالهتمام دون تقصير وإهمال لدراستك.

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