Dr. Pehrson. Oxidative phosphorylation. ATP Synthase enzyme Two functional parts, Fo intramembrane, F1 functional part, in matrix. 6 big subunits, kind of like orange segments. Has 3 ATP binding sites (one per dimer). Note that the three sites are NOT the same...diff conformation despite being made of same proteins. the tight binding site already has an ATP (binds ADP +P and forms ATP) but won't release it. The O site won't bind any ATP, and the low affinity L site will accept ADP + Pi. The proton flux through the protein causes a conformational change such that the T site becomes an O site, and releases the ATP, while the L site becomes the T site, and is able to make the ADP + P into ATP. So, these conformational changes are kind of a rotational movement, caused by the proton flux through the protein. The alpha beta dimers rotate around the gamma protein which is like the core. So the proton flux is used to drive the physical motion of these proteins. note discrepancy bet. handout and stryers. follow handout chart for # ATP per NADH and FADH2 ATP CYCLE ATP used for motion, active transport, biosyntheses, signal amplification, during which it is converted to ADP, which, by photosynthesis or oxidative phosphorylation is converted back to ATP. Half life of ATP is like a minute. The cycle goes very rapidly. You want these substances to be in some kind of balance - hopefully you don't use more ATP than you can produce! metabolism/activity of cells varies, so you want to be able to vary the cycle rate. You don't want to make too much extra ATP, or not enough. Energy charge = [ATP] + 1/2[ADP] ---------------- [ATP] + [ADP] +[AMP] measures amt in high energy state over amt total of adenosine nucleotides if everything is ATP, then your ratio is ONE. This never occurs. usually at about .8 or .9 Regulation: if there is no ADP around, ATP synthase can't do anything, and its proton channel will close. If the protons aren't flowing into matrix, proton gradient will continue to build up over inner membrane. At some point, as the gradient builds up, it's getting more and more difficult to pump protons accross the membrane, and at some point it becomes impossible to pump the protons into the intermembrane space and the electron transfer chain will slow and then stop. as ADP is exhausted, proton flow through atp synthase slows/stops, then proton pumping across gradient slows and stops, then ETC slows/stops, therefore O2 consumption slows/stops. when oxidative phosphorylation slows/stops, TCA cycle will slow/stop. when ETC slows, NADH -->NAD slows, so NADH builds up, and NAD level is low, and this will slow/stop the TCA cycle. muscle during exercise will have the energy charge drop, because you have less ATP relative to ADP, so there is more substrate for ATP synthase, so you will speed up Tight coupling between etc and atp synthase, but this can be interfered with. This can be done w/specific chemicals...2,4 Dinitrophenol, and carbonylcyanide p trifluoromethoxyphenylhydrazone. These chemicals are hydrophobic enough to penetrate membrane, and also have a proton that can be donated to the environment so can be ionized. So, if one of these moves into the mitochondrial matrix, it will disrupt the proton gradient, removing the energy needed to make ATP. So, in the presence of an uncoupler, the ETC keeps working very rapidly, because the gradient is being consumed, but the consumption isn't because of normal proton flow through ATP synthase, but is rather dissipating as heat, and no ATP is being produced. It's not a very healthy situation. But there are circumstances when it could be healthy...in BROWN FAT, which is full of mitochondria, and which is used to produce heat by burning itself, there is a special protein called uncoupling protein (thermogenin) which has a proton channel and diverts protons from ATPase, therefore uncoupling the reactions and generating heat. This is used widely in hibernating animals, animals in very could climates, and is present in most mammals to various extents. Maybe also involved in weight control. Mouse genetically engineered to have no brown fat...mouse became extremely obese on regular diet. So, it seems these cells may be used to regulate body weight. When atp synthesis is uncoupled from the ETC, it isn't TOTALLY uncoupled. Some of the protons still go through the ATP synthase, so you can generate low amounts of ATP this way, and perhaps also make some through glycolysis.. uncouplers won't make you "lose weight now" though. Most likely, you'd die ---symptoms include increased metabolism, fever, collapse, and death. brain would be most sensitive tissue, because is most oxygen dependent and most metabolically active. NADH which is made from glycolysis in the cytoplasm - electrons get into mitochondria Malate aspartate shuttle- oxaloacetate can be reduced to malate by NADH which was produced in cytoplasm. So the NADH becomes NAD and is recycled. Now the malate can go into the matrix of the mitochondria and can reduce the NAD there, oxidizing the malate back into oxaloacetate, which is then made into aspartate using aspartate aminotransferase. the aspartate can go back out to the intermembrane space and be used again. ??? NADH also used to reduce dihydroxyacetone phosphate to glycerol3phosphate, which is oxidized back into dihi.phosphate by FAD, which is reduced to FADH2 by glycerol3phosphate dehydrogenase. the FADH2 feeds electrons into the ETC at the point of compound Q (ubinquinone). ADP and phosphate also need to get into the mitochondria. ATP is made on the matrix side of the inner membrane, so you need substrate. So, adenosine nucleotide translocase can transport ATP and ADP across the inner membrane. Preferentially sends ADP in and ATP out due to electrochemical gradient. The outside of the membrane is +, and ATP is 4- while ADP is 3-,. so sending out an ATP and bringing in an ADP is electically favorable, dissipating a bit of the energy of the proton gradient. But, not that much energy lost. Phosphate goes into matrix through phospate translocase, also facilitated by proton gradient. The phosphate goes in with a proton. exact mechanism of proteins not well known. How is energy from proton gradient used? drives atp synthesis, active transport, energy source for bacterial flagellar rotation, heat production, NADPH synthesis. Major limitation of proton gradient vs ATP _ everything has to be localized across a membrane. ATP is soluble and can diffuse throughout the cell. Mitochondria and mitochondrial diseases. Usually several hundred mitochondria in a few cells. Mitochondria are kind of weird, have their own DNA, probably 'cause they used to be intracellular parasites. Mitochondrial DNA small, has been sequenced and is about 16000 base pairs. can encode only a few proteins - about 13 proteins, 22 TRNAs and 2 rRNAs. most of the proteins involved in mitochondrial function are encoded in the nucleus and then transported into the mitochondria. Mitochondria are almost entirely of maternal derivation - sperm have virtually no mitochondria. As cells grow and divide the mitochondria too have to grow and divide and replicate their dna. So, you basically have mom's mitochondria. mutation rate of mitochondrial DNA about 20x more than that in nucleus - likely due to oxidative environment and presence of superoxides. net result - mitochondrial genome accumulates damage over times, but there are plenty of reserves so there usually isn't a problem. genetic diseases of mitochondria show up about 40-50 yrs. in normal person, you can keep oxidative phosphorylation ok until about 100 yrs old, but if you have defective mitochondrial dna, you can have problems show up sooner, causing rapid onset blindness or other things.