as we were saying....ubiquinone is bound to the membrane in the mitochondria. Some people sell compound Q in health food stores.... So, the three protein complexes in the ETC (electron transport chain) are in the inner membrane of the mitochondria. recall that membranes are made of phospholipids, and will be discussed later. Remember there are two long fatty acid chains attached to glycerol which is attached to phosphate and serine. The phospholipis all have a polar head group and a hydrophobic hydrocarbon tail (the fatty acid chains.) So, they line up in membrane fashion to form a phospholipid bilayer. membranes have a relative impermeability to large, charged, polar substances, because the central part of the membrane is very hydrophobic. The lipid bilayer is very impermeable to Na+, K+, Cl-; is somewhat permeable o glucose, tryptophan, urea, glycerol; much more permeable to Indole and H2O. Now, the proteins used in the ETC are in the inner membrane of the mitochondria as stated above. Remember that some proteins form channels across the entire membrane, and others are partly or not at all embedded. The three large proteins used in ETC are transmembrane proteins. First complex of ETC is NADH dehydrogenase, which transfers electrons from NADH to ubiquinone. AKA NADH-Q reductase. This is a very large molecule with flavin nucleotide and iron-sulfur groups (?). This transfers the electrons to ubiquinone which then transfers electrons through the second complex, which is called cytochrome reductase, to cytochrome C, which uses cytochrome oxidase to transfer the electrons to molecular oxygen. note that also electrons can enter chain from FADH2 in flavoproteins, going directly to compound Q/ubiquinone, instead of coming from NADH directly. Reduction of O2 to H2O shown on overhead. not in handout, don't need to memorize, isn't proven. In the course of this theoretical process you produce an interesting molecule O2 + 1 e- -------> O2- (superoxide anion, a "free radical" with an unpaired e-) Free radicals are very reactive and are undesirable in biological systems. Because the cell doesn't want free radicals hanging around, they are very tightly bound by enzymes and not released into the cell. But, things don't go perfectly and so some of it will be released. so there are enzymes to take care of them Superoxide dismutase takes 2 O2- and makes H2O2 + O2 then, to break down the H2O2, you use catalase, which takes 2 H2O2 and forms 2H2O plus O2. There are a variety of peroxidases to break down other peroxides. antioxidant producers: activation of inflammatory cells xanthine oxidase disrupted mitochondrial electron transport cycooxygenase and lipoxygenase heme proteins no one sure what is most important free radical in terms of aging, cancer, tissue damage. people in some cases are promoting ingestion of antioxidants eg Vit E, C, beta carotene, to avoid such things as cancer etc. ALS (lou gehrig's dz) is caused by mutant superoxide dismutase which produces a product more destructive than the orginal free radical. chemoosmotic hypothesis: as electrons flow through the protein complexes, they pump protons from matrix into intermembrane space, creating proton gradient across the membrane. see cartoon top of p 7 in handout. so, part of the energy is conserved in form of proton gradient, because you have the energetically unfavored state of protons lining up on the outside, and you have the electric potential. in the process of synthesizing ATP, the protons go back into the matrix, dissipating the energy of the gradient. This whole process is oxidative phosphorylation: ETC and ATP synthesis. EVIDENCE for this model: Well, you can measure a pH gradient across the membrane, and there is a difference of about 1.4 pH units across the membrane, which is a pretty large proton gradient. This has sufficient energy to synthesize ATP with the transfer of 2 or 3 protons back across membrane. You could replace ETC with bacterial system utilizing light to create proton gradient, and you can then still make ATP. The inner membrane has to be sealed, because the proton gradient is vital. The protons, being charged, are not likely to get across membrane if it is in fact sealed. It appears that all 3 of the protein complexes participate in creation of the proton gradient. P/O ratio = # ATP/atom of O reduced. It takes 4 electrons to reduce molecular O2, so it takes 2 to reduce O. P/O ratio is different for diff substrates. For NADH, P/O ratio = 3/1 FADH2 has P/O ratio of 2/1. Ascorbate has P/O ratio of 1/1 (in vitro only.) How can you tell where substrates feed into the system?Britton Chance developed the CROSSOVER TECHNIQUE which depends on the ability to distinguish the oxidized and reduced forms of intermediates of the chain, as shown in graph on p.8 (they have specific wavelengths.) Two, it depends on existence of inhibitors. each step has an inhibitor that will block the transfer of e- at a particular step. If you add antimycin A to the ETC, you will have the earlier steps build up in a reduced state (electrons can't get past the block), whereas the later steps will be completely oxidized, with no new electrons flowing in. Say you block with amytal, and try to feed in with NADH - it won't work. but, if you try to feed the system with FADH2, it WILL work, because the block is BEFORE e- are fed into ubiquinone, and you can get 2 atp per atom of O being reduced. If you block w/antimycin A, FADH2 and NADH are BOTH unable to continue down pathway. How does ATP synthesis work?? This involves another large protein complex that traverses the inner membrane, known as ATP synthetase. The protein is made of 2 parts, F0 and F1. F1 is in the matrix doing the actual work, and F0 is in the membrane. Proton flux is coupled to ATP synthesis as shown on p.9 of handout. So, the enzyme has 3 binding sites for ATP. 0 site has low affinity, L site has a low affinity, and T site has a very high affinity for ATP. When this starts, T site already has an ATP in it. ADP + Pi add to the L site, and protons flow through the complex, changing the conformation of the sites. So, the former T site becomes a 0 site, releasing the ATP that was bound there. Then, the original L site becomes a T site synthesizing ATP from the ADP + Pi, and binds it so tightly that it can't release it. You need to have another proton flux through the protein, which will change the new-T site into an 0 site, and so forth. It would seem the protein is similar to a revolving door. final accounting of OP using glucose as an example. you get a net synthesis of +2 ATP from glycolysis in the mitochondria, you make 2 NADH from your 2 pyruvates from 1 glucose. citric acid cycle gets 2 GTP, 6 NADH, 2 FADH2 Oxidative phosphorylation the 2 NADH formed in glycolysis each yield 2 ATP (due to transport mechanism) other 2 NADH each yield 3 ATP 2 FADH2 each yield 2 ATP 6 NADH FROM CITRIC ACID CYCLE EACH YIELD 3 ATP. total ATP per glucose molecule = 36.