WEBVTT 00:00:00.090 --> 00:00:02.490 The following content is provided under a Creative 00:00:02.490 --> 00:00:04.030 Commons license. 00:00:04.030 --> 00:00:06.330 Your support will help MIT OpenCourseWare 00:00:06.330 --> 00:00:10.720 continue to offer high quality educational resources for free. 00:00:10.720 --> 00:00:13.320 To make a donation or view additional materials 00:00:13.320 --> 00:00:17.280 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.280 --> 00:00:18.450 at ocw.mit.edu. 00:00:21.000 --> 00:00:23.520 JOHN ESSIGMANN: Let's take a look at storyboard 14, 00:00:23.520 --> 00:00:25.500 where I discuss the Q cycle. 00:00:25.500 --> 00:00:28.320 If you look back at my previous lecture in which I introduced 00:00:28.320 --> 00:00:29.820 the respiratory apparatus, you'll 00:00:29.820 --> 00:00:32.430 see that there are three points in the electron transport 00:00:32.430 --> 00:00:34.620 chain, at which protons are translocated 00:00:34.620 --> 00:00:37.260 across the mitochondrial inner membrane, 00:00:37.260 --> 00:00:39.700 into the intermembrane space. 00:00:39.700 --> 00:00:44.070 The three sites are complex one, complex two, and complex four. 00:00:44.070 --> 00:00:46.140 If your entry point for the electrons 00:00:46.140 --> 00:00:48.810 is a pair of electrons at NADH, you'll 00:00:48.810 --> 00:00:51.460 translocate about 10 protons. 00:00:51.460 --> 00:00:53.670 This generates the electrochemical gradient 00:00:53.670 --> 00:00:58.530 that will be used in complex five, the F naught f1 synthase. 00:00:58.530 --> 00:01:01.650 And the energy released in proton translocation back 00:01:01.650 --> 00:01:06.187 into the cytoplasm, eventually will be used to synthesize ATP. 00:01:06.187 --> 00:01:07.770 At this point, I'd like to take a look 00:01:07.770 --> 00:01:10.170 at the hypothetical mechanism by which proton 00:01:10.170 --> 00:01:12.720 translocation into the intermembrane space 00:01:12.720 --> 00:01:15.150 happens in complex three. 00:01:15.150 --> 00:01:17.790 At the outset, let me say that the mechanism here 00:01:17.790 --> 00:01:20.070 is different from the mechanism at complex one 00:01:20.070 --> 00:01:21.150 and complex four. 00:01:21.150 --> 00:01:24.900 And I'll also mention that other organisms have found other ways 00:01:24.900 --> 00:01:27.900 to solve this problem of translocating protons 00:01:27.900 --> 00:01:30.730 into the intermembrane space. 00:01:30.730 --> 00:01:33.900 Let's look at panel A of storyboard 14. 00:01:33.900 --> 00:01:37.470 At the upper left, we see coenzyme Q in its quinone form. 00:01:37.470 --> 00:01:40.350 In this form, it's a taxadiene dione. 00:01:40.350 --> 00:01:41.815 It's one of our co-factors. 00:01:41.815 --> 00:01:44.760 And we've also referred to it in the past as a mobile electron 00:01:44.760 --> 00:01:45.840 carrier. 00:01:45.840 --> 00:01:48.720 In that regard, it falls into the same grouping of molecules, 00:01:48.720 --> 00:01:51.860 such as the NAD plus, NADH pair. 00:01:51.860 --> 00:01:53.460 The squiggly line at the lower right 00:01:53.460 --> 00:01:56.730 corner of the quinone molecule depicts isoprene units. 00:01:56.730 --> 00:01:59.040 Actually, there's a series of about six to 10 00:01:59.040 --> 00:02:01.650 of these isoprenes in the Q molecule. 00:02:01.650 --> 00:02:04.380 These probably facilitate association 00:02:04.380 --> 00:02:07.890 with hydrophobic regions, such as regions in membranes. 00:02:07.890 --> 00:02:10.350 The structure of coenzyme Q allows 00:02:10.350 --> 00:02:13.690 it to pick up electrons one at a time from an electron donor. 00:02:13.690 --> 00:02:15.601 And deliver them one at a time to a place 00:02:15.601 --> 00:02:17.100 where the electrons are going to go. 00:02:17.100 --> 00:02:19.620 That is an electron recipient. 00:02:19.620 --> 00:02:22.140 This panel shows the step-by-step mechanism 00:02:22.140 --> 00:02:24.240 by which electrons, one at a time, 00:02:24.240 --> 00:02:29.160 can reduce the oxidized form of coenzyme Q. Called Q here, 00:02:29.160 --> 00:02:34.230 or the quinone, to its fully reduced form QH2, also known 00:02:34.230 --> 00:02:35.610 as the hydroquinone. 00:02:35.610 --> 00:02:38.520 These electrons come from complex one, or complex two, 00:02:38.520 --> 00:02:41.550 or another membrane bound entry point for electrons 00:02:41.550 --> 00:02:43.500 into respiration. 00:02:43.500 --> 00:02:47.640 The first electron converts the quinone Q to the semiquinone 00:02:47.640 --> 00:02:48.690 radical. 00:02:48.690 --> 00:02:51.870 The semiquinone will then pick up another proton and electron 00:02:51.870 --> 00:02:55.050 to form the fully reduced species, the hydroquinone, 00:02:55.050 --> 00:02:57.020 or QH2. 00:02:57.020 --> 00:03:00.920 The hydroquinone QH2 is fully loaded with electrons. 00:03:00.920 --> 00:03:03.740 The fully reduced hydroquinone will next deliver its two 00:03:03.740 --> 00:03:06.260 electrons to complex three. 00:03:06.260 --> 00:03:08.000 The technical name of complex three 00:03:08.000 --> 00:03:12.500 is coenzyme Q, cytochrome c oxidoreductase. 00:03:12.500 --> 00:03:14.960 This name gives a hint that the electrons 00:03:14.960 --> 00:03:18.680 are going to pass from coenzyme Q in its reduced form, 00:03:18.680 --> 00:03:21.890 to an oxidized form of cytochrome c, which 00:03:21.890 --> 00:03:26.360 is also non-covalently associated with complex three. 00:03:26.360 --> 00:03:30.140 Let's look at panel B. A single molecule of QH2, 00:03:30.140 --> 00:03:32.720 the hydroquinone, contains two electrons that 00:03:32.720 --> 00:03:35.480 are going to be transferred to complex three. 00:03:35.480 --> 00:03:38.920 One electron is going to reduce iron three to iron two, 00:03:38.920 --> 00:03:41.559 in a first molecule of cytochrome C. 00:03:41.559 --> 00:03:43.100 And then the second electron is going 00:03:43.100 --> 00:03:47.240 to be used to reduce a second molecule of cytochrome c. 00:03:47.240 --> 00:03:49.730 It's convenient to break this overall reaction 00:03:49.730 --> 00:03:50.880 scheme into two parts. 00:03:50.880 --> 00:03:53.020 I'll call them cycle one and cycle two. 00:03:53.020 --> 00:03:55.940 In cycle one, the first molecule of cytochrome C 00:03:55.940 --> 00:03:57.380 is going to be reduced. 00:03:57.380 --> 00:03:59.720 And then in cycle two, the second molecule 00:03:59.720 --> 00:04:01.900 will be reduced. 00:04:01.900 --> 00:04:06.130 I've further broken down the cycle into eight steps. 00:04:06.130 --> 00:04:08.140 At the upper left of complex three, 00:04:08.140 --> 00:04:10.480 you'll see a site that I've marked with an x. 00:04:10.480 --> 00:04:12.910 This is an iron cell for protein. 00:04:12.910 --> 00:04:17.410 This is going to be that docking site for QH2, the semiquinone. 00:04:17.410 --> 00:04:19.959 Sometimes it is called the Q delta site. 00:04:19.959 --> 00:04:24.910 The reduced quinone, QH2, gives up two protons and one electron 00:04:24.910 --> 00:04:28.450 to form the semiquinone radical, Q.minus. 00:04:28.450 --> 00:04:30.610 The electron in step three goes over 00:04:30.610 --> 00:04:33.430 to reduce ferric ion to ferrous iron, that 00:04:33.430 --> 00:04:37.180 is iron three to iron two in cytochrome c. 00:04:37.180 --> 00:04:39.910 As I mentioned earlier, cytochrome c 00:04:39.910 --> 00:04:43.060 is another one of our mobile electron carriers. 00:04:43.060 --> 00:04:46.660 It's going to float away and go off to complex four. 00:04:46.660 --> 00:04:49.900 At this point in step four B, the semiquinone radical 00:04:49.900 --> 00:04:52.000 is going to give up its second electron 00:04:52.000 --> 00:04:56.110 to cytochrome bL, which is a constituent 00:04:56.110 --> 00:04:58.300 of the complex three. 00:04:58.300 --> 00:05:01.180 From the standpoint of coenzyme Q, at this point 00:05:01.180 --> 00:05:03.610 it's lost both of its electrons. 00:05:03.610 --> 00:05:06.190 It has been converted to the fully oxidized form 00:05:06.190 --> 00:05:10.360 Q, as depicted in step four A. The electron 00:05:10.360 --> 00:05:13.740 at cytochrome bL flows into cytochrome bH. 00:05:13.740 --> 00:05:15.310 And from there, it will subsequently 00:05:15.310 --> 00:05:20.770 flow into a molecule of the parental diquinone Q. The one 00:05:20.770 --> 00:05:23.920 electron reduction of the diquinone Q, results 00:05:23.920 --> 00:05:26.440 in the formation of the semiquinone radical, 00:05:26.440 --> 00:05:28.230 once again. 00:05:28.230 --> 00:05:30.090 This is at position Y-- 00:05:30.090 --> 00:05:34.980 the way I've drawn the complex three in panel B. Position Y 00:05:34.980 --> 00:05:38.140 is also called the Qi site. 00:05:38.140 --> 00:05:40.360 At this point, the semiquinone radical 00:05:40.360 --> 00:05:42.610 will just stay where it is for a few minutes. 00:05:42.610 --> 00:05:46.540 We'll pick it up again in the second half of the Q Cycle. 00:05:46.540 --> 00:05:49.570 To summarize what happened in the first half of the Q Cycle, 00:05:49.570 --> 00:05:52.810 two protons have been translocated from the matrix 00:05:52.810 --> 00:05:54.910 into the intermembrane space. 00:05:54.910 --> 00:05:56.500 And one electron has been transferred 00:05:56.500 --> 00:06:00.250 to cytochrome c, which then translocates 00:06:00.250 --> 00:06:03.580 across the outer surface of the mitochondrial inner membrane 00:06:03.580 --> 00:06:05.980 to complex four. 00:06:05.980 --> 00:06:08.470 At this point, please look at panel C. 00:06:08.470 --> 00:06:11.070 Now we're going to take a look at the second half of the Q 00:06:11.070 --> 00:06:12.040 Cycle. 00:06:12.040 --> 00:06:14.200 At the bottom of complex three in step 1, 00:06:14.200 --> 00:06:16.600 you'll see the orphaned semiquinone radical 00:06:16.600 --> 00:06:17.710 that we just created. 00:06:17.710 --> 00:06:20.200 Keep in mind that we are going to come back 00:06:20.200 --> 00:06:22.070 and use that radical in a couple of minutes. 00:06:22.070 --> 00:06:23.290 So bear with me. 00:06:23.290 --> 00:06:25.780 At step two, we see a second molecule 00:06:25.780 --> 00:06:28.850 of the hydroquinone, QH2, inside the mitochondrial inner 00:06:28.850 --> 00:06:29.860 membrane. 00:06:29.860 --> 00:06:32.830 We're going to borrow this molecule for a short time. 00:06:32.830 --> 00:06:34.480 And then we're going to restore it. 00:06:34.480 --> 00:06:37.070 So this QH2 is, essentially, catalytic 00:06:37.070 --> 00:06:39.280 in the overall reaction scheme. 00:06:39.280 --> 00:06:42.340 In step three, we see that this borrowed molecule 00:06:42.340 --> 00:06:47.080 of the hydroquinone, QH2, gives up two protons and one electron 00:06:47.080 --> 00:06:50.960 just as we had seen in the first half of the cycle. 00:06:50.960 --> 00:06:52.450 You will note that we are reducing 00:06:52.450 --> 00:06:56.200 a second molecule of cytochrome c, which is then going 00:06:56.200 --> 00:06:58.020 to go off to complex four. 00:06:58.020 --> 00:07:00.550 And we're also going to be producing 00:07:00.550 --> 00:07:03.850 a second molecule of coenzyme Q, the fully 00:07:03.850 --> 00:07:06.840 oxidized form of the co-factor. 00:07:06.840 --> 00:07:09.970 In step four B, the semiquinone radical, just as 00:07:09.970 --> 00:07:12.090 happened in cycle one, will give up 00:07:12.090 --> 00:07:14.650 its electron to cytochrome bL. 00:07:14.650 --> 00:07:17.350 The electron will then go to cytochrome bH, 00:07:17.350 --> 00:07:20.650 and then will flow into the semiquinone radical 00:07:20.650 --> 00:07:23.740 that we had left over from the first cycle. 00:07:23.740 --> 00:07:27.310 This radical exists at site Y, or as I called it before, 00:07:27.310 --> 00:07:30.190 the Qi site of complex three. 00:07:30.190 --> 00:07:32.800 The reduction of the semiquinone radical at step six 00:07:32.800 --> 00:07:36.160 is concomitant in step seven with the acquisition 00:07:36.160 --> 00:07:38.680 of two protons from the matrix. 00:07:38.680 --> 00:07:41.020 This series of reactions, ultimately, 00:07:41.020 --> 00:07:45.030 results in restoration of QH2, the semiquinone 00:07:45.030 --> 00:07:46.960 in the mitochondrial inner membrane. 00:07:46.960 --> 00:07:49.630 At this point, we have restored the molecule of QH2, 00:07:49.630 --> 00:07:51.850 at step eight, that we had borrowed, initially, 00:07:51.850 --> 00:07:53.260 at step two. 00:07:53.260 --> 00:07:55.210 The second cycle has also produced 00:07:55.210 --> 00:07:58.870 net Q, the oxidized form of the co-factor, 00:07:58.870 --> 00:08:02.430 which is now free to go on to complexes one and two, 00:08:02.430 --> 00:08:04.990 and to pick up more reducing equivalents. 00:08:04.990 --> 00:08:07.990 Just looking at the second half of the Q Cycle, what we see 00:08:07.990 --> 00:08:10.870 is that we've translocated another two protons 00:08:10.870 --> 00:08:12.850 into the intermembrane space. 00:08:12.850 --> 00:08:16.900 And we have transferred a second electron to cytochrome c. 00:08:16.900 --> 00:08:20.500 So cycle one and cycle two, each, 00:08:20.500 --> 00:08:22.990 result in the translocation of two protons 00:08:22.990 --> 00:08:25.240 from the matrix to the intermembrane space. 00:08:25.240 --> 00:08:28.090 And each results in the transfer of one electron 00:08:28.090 --> 00:08:29.770 to cytochrome c. 00:08:29.770 --> 00:08:33.130 Looking at the whole Q Cycle, that is cycle one and cycle two 00:08:33.130 --> 00:08:37.539 together, we get a pair of electrons that enter from QH2. 00:08:37.539 --> 00:08:41.320 Four protons are translocated into the intermembrane space. 00:08:41.320 --> 00:08:44.140 And two reduced molecules of cytochrome c, 00:08:44.140 --> 00:08:46.510 which then migrate to complex four, 00:08:46.510 --> 00:08:49.420 where they'll be later oxidized. 00:08:49.420 --> 00:08:54.580 Let's turn to storyboard 15 and panel A. In panel A, 00:08:54.580 --> 00:08:58.050 I'm showing another way to look at the Q Cycle. 00:08:58.050 --> 00:09:00.340 I'll emphasize that this is not as chemically 00:09:00.340 --> 00:09:04.290 accurate as the two step process that I showed you previously. 00:09:04.290 --> 00:09:06.670 And I'm not going to discuss it here further. 00:09:06.670 --> 00:09:08.740 Nevertheless, I think that this presentation 00:09:08.740 --> 00:09:10.810 makes it relatively easy for you to see 00:09:10.810 --> 00:09:14.850 the overall stoichiometry of the Q Cycle. 00:09:14.850 --> 00:09:18.450 Let's now turn to storyboard 15, panel B. 00:09:18.450 --> 00:09:22.000 In the way of a high-level review of electron transport 00:09:22.000 --> 00:09:25.650 so far, electrons from nutrient oxidation 00:09:25.650 --> 00:09:29.670 have been deposited into the electron transport chain. 00:09:29.670 --> 00:09:32.190 The transfer of electrons to oxygen 00:09:32.190 --> 00:09:35.490 resulted in energy that is used to power pumps-- 00:09:35.490 --> 00:09:39.480 pumps that pump protons into the intermembrane space. 00:09:39.480 --> 00:09:42.480 We've looked at the Q Cycle as one example, of several, 00:09:42.480 --> 00:09:44.970 of how protons are pumped. 00:09:44.970 --> 00:09:47.760 It took energy to create this proton gradient. 00:09:47.760 --> 00:09:49.410 When we release the proton gradient 00:09:49.410 --> 00:09:53.870 by allowing the protons to flow through the ATP synthase, 00:09:53.870 --> 00:09:56.040 we're going to be able to use that energy in order 00:09:56.040 --> 00:09:58.740 to accomplish the otherwise energetically 00:09:58.740 --> 00:10:03.030 uphill phosphorylation of ADP into ATP. 00:10:03.030 --> 00:10:06.720 Now let's look at storyboard 15, panel C. This panel 00:10:06.720 --> 00:10:11.190 shows the F naught F1 proton translocating ATP synthase, 00:10:11.190 --> 00:10:13.770 also known as the ATP synthase. 00:10:13.770 --> 00:10:16.020 To put things in perspective, at the top 00:10:16.020 --> 00:10:18.210 is the intermembrane space, which 00:10:18.210 --> 00:10:20.910 is where the protons have been translocated. 00:10:20.910 --> 00:10:23.610 In the middle is the mitochondrial inner membrane. 00:10:23.610 --> 00:10:26.460 And at the bottom is the mitochondrial matrix. 00:10:26.460 --> 00:10:30.380 Because we've pumped protons into the intermembrane space, 00:10:30.380 --> 00:10:33.030 its pH is about three quarters of a pH unit 00:10:33.030 --> 00:10:36.810 lower than the pH of the matrix of the mitochondria. 00:10:36.810 --> 00:10:39.510 Proton flow is going to be regulated in response 00:10:39.510 --> 00:10:41.730 to physiologic needs. 00:10:41.730 --> 00:10:45.610 I'll talk about that regulation and how it occurs later. 00:10:45.610 --> 00:10:49.200 For now, however, let's look at protons flowing through the F 00:10:49.200 --> 00:10:50.850 naught F1 complex. 00:10:50.850 --> 00:10:52.740 Let's imagine that the broken line 00:10:52.740 --> 00:10:56.286 indicates a channel, through which protons will flow. 00:10:56.286 --> 00:10:57.660 Other structural features include 00:10:57.660 --> 00:11:00.480 a shaft, which I've indicated as gamma, 00:11:00.480 --> 00:11:03.990 and a ring, in which the shaft is embedded, 00:11:03.990 --> 00:11:06.180 which I've indicated as the C-ring. 00:11:06.180 --> 00:11:09.030 When protons flow, both the shaft and the C-ring 00:11:09.030 --> 00:11:13.690 will move, as you'll see, in a clockwise rotation. 00:11:13.690 --> 00:11:15.400 At the bottom of the overall apparatus 00:11:15.400 --> 00:11:18.790 are three alpha and three beta subunits. 00:11:18.790 --> 00:11:20.530 These are not going to rotate. 00:11:20.530 --> 00:11:22.270 That is, they're going to stay fixed 00:11:22.270 --> 00:11:26.272 in position while the C-ring and the gamma subunit rotate. 00:11:26.272 --> 00:11:29.050 The beta subunit contains a site x, 00:11:29.050 --> 00:11:32.080 which is going to be the active site for conversion of ADP 00:11:32.080 --> 00:11:34.960 plus inorganic phosphate to ATP. 00:11:34.960 --> 00:11:38.890 I've drawn a little elbow on the bottom of the gamma subunit. 00:11:38.890 --> 00:11:41.080 Let's imagine that protons are flowing. 00:11:41.080 --> 00:11:43.720 And the flow makes that subunit spin around. 00:11:43.720 --> 00:11:45.670 And further, imagine that the elbow is 00:11:45.670 --> 00:11:48.040 bumping into the active sites-- 00:11:48.040 --> 00:11:49.420 that is, the x sites. 00:11:49.420 --> 00:11:51.370 There are three of the x sites, one 00:11:51.370 --> 00:11:53.230 on each of the b subunits at the bottom 00:11:53.230 --> 00:11:55.180 of the overall apparatus. 00:11:55.180 --> 00:11:59.500 Imagine that the energy involved is translocated from the elbow 00:11:59.500 --> 00:12:01.150 to the active sites. 00:12:01.150 --> 00:12:05.050 And that's what's going to help us align ADP and Pi, 00:12:05.050 --> 00:12:08.920 to make the overall chemical reaction favorable. 00:12:08.920 --> 00:12:11.260 That is a hypothetical, but not far fetched way 00:12:11.260 --> 00:12:15.010 to think about the way that ATP is made. 00:12:15.010 --> 00:12:18.190 Let's now look at panel D. The current view 00:12:18.190 --> 00:12:21.160 is that the active site oscillates among three 00:12:21.160 --> 00:12:23.260 different conformations during the time 00:12:23.260 --> 00:12:25.300 the protons are translocated. 00:12:25.300 --> 00:12:27.070 These three conformational states 00:12:27.070 --> 00:12:31.630 are referred to as O for open, L for loose, and T for tight. 00:12:31.630 --> 00:12:35.260 In the open state, nothing is present at the active site x. 00:12:35.260 --> 00:12:37.240 The conversion of open to loose is 00:12:37.240 --> 00:12:41.620 concomitant with the binding of ADP and inorganic phosphate. 00:12:41.620 --> 00:12:44.200 As protons continue to flow, the loose site 00:12:44.200 --> 00:12:46.840 is converted into the tight state. 00:12:46.840 --> 00:12:50.020 The energy associated with this conformational change results 00:12:50.020 --> 00:12:52.810 in the alignment of the inorganic phosphate, 00:12:52.810 --> 00:12:54.850 such that the conditions are now favorable 00:12:54.850 --> 00:12:58.300 for it to phosphorylate ADP and form ATP. 00:12:58.300 --> 00:13:01.780 Further proton flow results in the tight state being converted 00:13:01.780 --> 00:13:05.620 to the open state, which ejects the formed ATP out 00:13:05.620 --> 00:13:08.090 into the mitochondrial matrix. 00:13:08.090 --> 00:13:12.790 So at each cycle, of open to loose to tight, 00:13:12.790 --> 00:13:14.980 a molecule of ATP is made. 00:13:14.980 --> 00:13:16.780 And then it is ejected. 00:13:16.780 --> 00:13:19.520 This goes on again, and again, and again. 00:13:19.520 --> 00:13:23.580 That's the overall mechanism by which ATP is synthesized. 00:13:23.580 --> 00:13:26.970 At the high level, proton flow results in the movement 00:13:26.970 --> 00:13:28.420 as the turning of a rotor. 00:13:28.420 --> 00:13:30.000 It is a lot like a water wheel that 00:13:30.000 --> 00:13:33.030 might drive the rotation of a millstone, that grinds grain 00:13:33.030 --> 00:13:34.530 into flour. 00:13:34.530 --> 00:13:36.840 The rotation of the shaft provides energy 00:13:36.840 --> 00:13:38.970 to bump into the x sites, resulting 00:13:38.970 --> 00:13:41.160 in the conversion of a first subunit 00:13:41.160 --> 00:13:44.280 to the open conformation, another subunit 00:13:44.280 --> 00:13:49.290 to the loose state, and a third subunit to tight. 00:13:49.290 --> 00:13:50.970 Then the process starts over again, 00:13:50.970 --> 00:13:55.050 with each cycle producing one molecule of ATP. 00:13:55.050 --> 00:13:56.190 As one final point-- 00:13:56.190 --> 00:13:59.880 a point that explains how this process is regulated. 00:13:59.880 --> 00:14:03.300 Proton flow results from the binding of ADP-- 00:14:03.300 --> 00:14:05.310 that is, ADP. 00:14:05.310 --> 00:14:07.410 This will become important in a few minutes 00:14:07.410 --> 00:14:11.130 when we talk about how the whole system is regulated. 00:14:11.130 --> 00:14:13.620 Now let's look at storyboard 16. 00:14:13.620 --> 00:14:16.560 Let's look at all four panels, panels A through D. 00:14:16.560 --> 00:14:18.070 In the last part of this lecture, 00:14:18.070 --> 00:14:20.010 I'd like to talk about how respiration 00:14:20.010 --> 00:14:23.280 is coupled to the TCA cycle and glycolysis. 00:14:23.280 --> 00:14:25.314 This is really the first time in 5.07 00:14:25.314 --> 00:14:26.730 that we're going to see the beauty 00:14:26.730 --> 00:14:29.250 of coordinated pathway regulation. 00:14:29.250 --> 00:14:32.190 I'm going to use a physiological scenario in order, 00:14:32.190 --> 00:14:34.909 hopefully, to make it all make sense. 00:14:34.909 --> 00:14:37.200 In this scenario, imagine that you're being chased down 00:14:37.200 --> 00:14:39.120 the street by a dog. 00:14:39.120 --> 00:14:41.310 In order to start running away from the dog, 00:14:41.310 --> 00:14:44.490 we're going to need some ATP that's very quickly generated 00:14:44.490 --> 00:14:46.930 from glucose by glycolysis. 00:14:46.930 --> 00:14:50.040 The TCA cycle is also going to be operative, 00:14:50.040 --> 00:14:52.330 along with a pathway we haven't seen so far, 00:14:52.330 --> 00:14:54.090 fatty acid oxidation. 00:14:54.090 --> 00:14:57.930 In both cases, the TCA cycle and fatty acid oxidation, 00:14:57.930 --> 00:15:00.580 reduced electron carriers are going to be produced. 00:15:00.580 --> 00:15:03.150 They're going to give up their electrons to the electron 00:15:03.150 --> 00:15:04.410 transport chain. 00:15:04.410 --> 00:15:08.910 Ultimately, to reduce oxygen to water and with concomitant 00:15:08.910 --> 00:15:11.860 pumping of protons into the intermembrane space, 00:15:11.860 --> 00:15:13.590 resulting in the lowering of the pH-- 00:15:13.590 --> 00:15:17.890 that is, acidification of the intermembrane space. 00:15:17.890 --> 00:15:19.690 As I mentioned earlier, that flow 00:15:19.690 --> 00:15:21.640 of protons through the ATP synthase 00:15:21.640 --> 00:15:23.620 is triggered by the binding of ADP 00:15:23.620 --> 00:15:27.587 to the x site on the beta subunit of the enzyme complex. 00:15:27.587 --> 00:15:29.920 As your muscles are working hard and you're running away 00:15:29.920 --> 00:15:32.200 from the dog, the concentration of ADP 00:15:32.200 --> 00:15:35.170 in the mitochondrial matrix is going to be increasing. 00:15:35.170 --> 00:15:38.650 Thus, proton flow is going to be initiated. 00:15:38.650 --> 00:15:40.870 As you run more and more, the protons 00:15:40.870 --> 00:15:43.900 are depleted from the intermembrane space. 00:15:43.900 --> 00:15:47.500 It's not known exactly how this reduction in proton 00:15:47.500 --> 00:15:49.960 concentration results in accelerated 00:15:49.960 --> 00:15:53.320 movement of electrons through the electron transport chain. 00:15:53.320 --> 00:15:56.050 It's tempting to speculate that one or more 00:15:56.050 --> 00:15:59.170 of the reductase centers, within the electron transfer chain, 00:15:59.170 --> 00:16:02.300 may be pH sensitive. 00:16:02.300 --> 00:16:04.420 Let's imagine that that is the case. 00:16:04.420 --> 00:16:07.030 So let's imagine that the raising of pH-- 00:16:07.030 --> 00:16:10.300 that is, the decrease in hydrogen ion concentration 00:16:10.300 --> 00:16:12.730 in the intermembrane space, results 00:16:12.730 --> 00:16:16.180 in the facilitated flow of electrons from complex one 00:16:16.180 --> 00:16:18.670 or complex two, to oxygen. 00:16:18.670 --> 00:16:23.500 Next, let's look to the far left at the bottom of panel D. 00:16:23.500 --> 00:16:26.740 As you draw more electrons into respiration, 00:16:26.740 --> 00:16:30.520 NADH is going to be converted to NAD plus. 00:16:30.520 --> 00:16:34.600 It's important, now, to remember that NADH feedback inhibits 00:16:34.600 --> 00:16:38.070 any step at which it's produced in the TCA cycle. 00:16:38.070 --> 00:16:40.450 Thus, as you're running away from the dog, 00:16:40.450 --> 00:16:43.396 the NADH concentration drops. 00:16:43.396 --> 00:16:44.770 And that means that there's going 00:16:44.770 --> 00:16:48.110 to be less inhibition of the steps at which NADH 00:16:48.110 --> 00:16:51.340 is produced in the TCA cycle. 00:16:51.340 --> 00:16:54.640 Thus, looking at the big picture, the binding 00:16:54.640 --> 00:16:59.260 of increased concentrations of ADP to the ATP synthase, 00:16:59.260 --> 00:17:02.840 over on the far right, results in the activation of the TCA 00:17:02.840 --> 00:17:05.260 cycle and other metabolic pathways that 00:17:05.260 --> 00:17:07.299 will result in the delivery of more electrons 00:17:07.299 --> 00:17:09.040 through the electron transport chain, 00:17:09.040 --> 00:17:10.900 in order to allow you to continue 00:17:10.900 --> 00:17:12.750 to run away from the dog. 00:17:12.750 --> 00:17:16.720 Next, let's imagine that you've been running for quite a while. 00:17:16.720 --> 00:17:20.200 And let's look at complex four, where oxygen is bound. 00:17:20.200 --> 00:17:23.470 Sooner or later you're going to be becoming oxygen limited. 00:17:23.470 --> 00:17:25.329 You're going to be panting heavily. 00:17:25.329 --> 00:17:28.300 This means that electron flow from a reduced electron 00:17:28.300 --> 00:17:31.450 carriers to oxygen is going to slow down. 00:17:31.450 --> 00:17:35.590 As we become more hypoxic, respiration starts to fail, 00:17:35.590 --> 00:17:38.470 but hypoxia dramatically increases 00:17:38.470 --> 00:17:41.740 the levels or activities of the enzymes of glycolysis. 00:17:41.740 --> 00:17:43.990 Thus, glycolysis will switch over 00:17:43.990 --> 00:17:47.740 to becoming our principal ATP generation machine. 00:17:47.740 --> 00:17:49.820 As we increase the rate of glycolysis, 00:17:49.820 --> 00:17:52.360 the flux goes from glucose to pyruvate, 00:17:52.360 --> 00:17:54.880 but the pyruvate can't be oxidized 00:17:54.880 --> 00:17:56.650 because it can't enter respiration, 00:17:56.650 --> 00:17:59.290 because we don't have enough oxygen present in order 00:17:59.290 --> 00:18:01.490 to oxidize it. 00:18:01.490 --> 00:18:04.720 So what happens is we activate lactate dehydrogenase 00:18:04.720 --> 00:18:07.300 to convert pyruvate to lactate. 00:18:07.300 --> 00:18:10.790 Conversion of pyruvate to lactate has two consequences. 00:18:10.790 --> 00:18:13.480 The first is that conversion results in the conversion 00:18:13.480 --> 00:18:15.310 of NADH to NAD plus. 00:18:15.310 --> 00:18:18.190 Remember, that this is the lactate shuttle. 00:18:18.190 --> 00:18:21.000 And that NAD plus is now available to allow 00:18:21.000 --> 00:18:23.260 glyceraldehyde 3-phosphate dehydrogenase 00:18:23.260 --> 00:18:28.030 to continue processing molecules of glucose into ATP. 00:18:28.030 --> 00:18:29.830 A second consequence of activation 00:18:29.830 --> 00:18:31.630 of lactate dehydrogenase is the fact 00:18:31.630 --> 00:18:34.540 that lactate spills out into the blood, where 00:18:34.540 --> 00:18:35.940 it acidifies the blood. 00:18:35.940 --> 00:18:37.390 The pH drops. 00:18:37.390 --> 00:18:40.150 Now let's think about what the consequences are 00:18:40.150 --> 00:18:42.610 when the pH of the blood drops. 00:18:42.610 --> 00:18:45.520 I want you to think back to the lectures in which JoAnne talked 00:18:45.520 --> 00:18:48.880 about cooperatively, the Bohr effect, and the affinity 00:18:48.880 --> 00:18:50.730 of oxygen for hemoglobin. 00:18:50.730 --> 00:18:54.910 JoAnne told us that protons are heterotropic allosteric 00:18:54.910 --> 00:18:58.390 effectors that, effectively, loosen the affinity of oxygen 00:18:58.390 --> 00:18:59.890 for hemoglobin. 00:18:59.890 --> 00:19:02.950 So as you're running away from the dog, 00:19:02.950 --> 00:19:05.920 glycolysis becomes the main pathway. 00:19:05.920 --> 00:19:09.040 You produce a lot of lactate that goes into the blood. 00:19:09.040 --> 00:19:10.570 The pH drops. 00:19:10.570 --> 00:19:13.390 Oxygen lowers its affinity for hemoglobin, 00:19:13.390 --> 00:19:17.800 and thus, becomes more available for the pathway of respiration. 00:19:17.800 --> 00:19:22.150 Oxygen is now back in the system and binds to complex four. 00:19:22.150 --> 00:19:25.930 So you kind of get what we could call a second wind that 00:19:25.930 --> 00:19:28.690 allows you to continue using respiration 00:19:28.690 --> 00:19:30.490 to run away from the dog. 00:19:30.490 --> 00:19:33.520 In other words, you've adapted to the stressful state 00:19:33.520 --> 00:19:37.330 and are now able to continue running. 00:19:37.330 --> 00:19:39.760 Now let's put it all together. 00:19:39.760 --> 00:19:41.260 A dog starts chasing you. 00:19:41.260 --> 00:19:44.140 Your readily available energy reserves, and free glucose, 00:19:44.140 --> 00:19:45.640 as well as glycogen, will produce 00:19:45.640 --> 00:19:49.360 rapid ATP that will get you moving away from the dog. 00:19:49.360 --> 00:19:52.720 Additionally, respiration will be contributing lots of ATP 00:19:52.720 --> 00:19:54.640 to allow you to escape the dog. 00:19:54.640 --> 00:19:57.640 And as you're running, ADP concentrations 00:19:57.640 --> 00:19:59.980 will go up inside your mitochondria. 00:19:59.980 --> 00:20:03.160 That will, basically, release the proton gradient, 00:20:03.160 --> 00:20:07.120 allowing the ATP synthase to work very quickly and very hard 00:20:07.120 --> 00:20:09.740 to make a lot more ATP. 00:20:09.740 --> 00:20:12.310 NADH concentrations in the mitochondria 00:20:12.310 --> 00:20:14.710 will drop with physical activities. 00:20:14.710 --> 00:20:18.250 This helps boot up the dehydrogenases of the TCA 00:20:18.250 --> 00:20:18.820 cycle. 00:20:18.820 --> 00:20:20.830 They become less inhibited. 00:20:20.830 --> 00:20:23.050 Your TCA cycle will accelerate, in order 00:20:23.050 --> 00:20:25.690 to produce more reducing equivalents to power 00:20:25.690 --> 00:20:28.030 the electron transport chain. 00:20:28.030 --> 00:20:31.750 All of this will continue until your oxygen becomes limiting, 00:20:31.750 --> 00:20:37.120 then respiration starts to fail, but the hypoxic state turns up 00:20:37.120 --> 00:20:39.880 the activities of the enzymes of glycolysis, 00:20:39.880 --> 00:20:44.230 thus glycolysis accelerates and helps accommodate. 00:20:44.230 --> 00:20:46.810 But pyruvate, at the end of glycolysis, 00:20:46.810 --> 00:20:49.570 cannot be further oxidized because there's not enough 00:20:49.570 --> 00:20:53.060 oxygen. It has to be converted to lactate. 00:20:53.060 --> 00:20:55.420 The lactate acidifies the blood. 00:20:55.420 --> 00:20:58.240 The Bohr effect causes the release of more oxygen 00:20:58.240 --> 00:21:01.720 into the blood, making it more available to complex four, 00:21:01.720 --> 00:21:04.840 in order to reboot the electron transport chain. 00:21:04.840 --> 00:21:08.410 Overall, this is a marvelously regulated physiological system 00:21:08.410 --> 00:21:12.240 that seems to have evolved in order to promote our survival.