1 00:00:00,500 --> 00:00:02,830 The following content is provided under a Creative 2 00:00:02,830 --> 00:00:04,370 Commons license. 3 00:00:04,370 --> 00:00:06,670 Your support will help MIT OpenCourseWare 4 00:00:06,670 --> 00:00:11,030 continue to offer high quality educational resources for free. 5 00:00:11,030 --> 00:00:13,660 To make a donation or view additional materials 6 00:00:13,660 --> 00:00:17,610 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,610 --> 00:00:18,540 at ocw.mit.edu. 8 00:00:25,760 --> 00:00:30,690 PROFESSOR: What I want to do is proceed where we left off. 9 00:00:30,690 --> 00:00:34,950 We're in module seven on reactive oxygen species. 10 00:00:34,950 --> 00:00:37,860 I'm introducing you to the concept, 11 00:00:37,860 --> 00:00:39,690 what was the big picture. 12 00:00:39,690 --> 00:00:42,920 And at the end of the last lecture, 13 00:00:42,920 --> 00:00:44,970 I gave you a few introductory slides, 14 00:00:44,970 --> 00:00:47,680 but what we're going to be focusing on, 15 00:00:47,680 --> 00:00:54,900 I told you was oxygen can be one electron reduced to superoxide. 16 00:00:54,900 --> 00:00:58,170 Superoxide can pick up another electron. 17 00:00:58,170 --> 00:01:00,450 They should have a couple protons here 18 00:01:00,450 --> 00:01:02,080 to form hydrogen peroxide. 19 00:01:02,080 --> 00:01:05,400 So these are two of the species we'll be looking at. 20 00:01:05,400 --> 00:01:09,270 If you have iron 2 around, so this goes back 21 00:01:09,270 --> 00:01:11,580 to the connection to iron homeostasis 22 00:01:11,580 --> 00:01:14,280 that we talked about in module 6, 23 00:01:14,280 --> 00:01:16,980 this is called Fenton's chemistry. 24 00:01:16,980 --> 00:01:18,810 And you produce hydroxide radicals, 25 00:01:18,810 --> 00:01:22,020 so that's the third reactive species. 26 00:01:22,020 --> 00:01:24,300 And hydrogen peroxide in the presence 27 00:01:24,300 --> 00:01:27,570 of chloride with myeloperoxidase can 28 00:01:27,570 --> 00:01:30,840 form hypochlorous acid, which then 29 00:01:30,840 --> 00:01:33,930 can chlorinate amino acids or sugars 30 00:01:33,930 --> 00:01:36,040 or a lot of other things. 31 00:01:36,040 --> 00:01:39,250 So you get rampant chlorination inside the cells. 32 00:01:39,250 --> 00:01:44,370 So this cartoon also has reactive nitrogen species. 33 00:01:44,370 --> 00:01:48,690 We're only focusing on the reactive oxygen species. 34 00:01:48,690 --> 00:01:52,710 So that's what we're going. 35 00:01:52,710 --> 00:01:58,180 And so at the beginning of the lecture, 36 00:01:58,180 --> 00:01:59,870 I wanted to introduce you-- 37 00:01:59,870 --> 00:02:02,830 and I did give you an overview of where we were going. 38 00:02:02,830 --> 00:02:05,730 We were going to look at what are the reactive oxygen 39 00:02:05,730 --> 00:02:10,500 species, what does reactivity mean, how do you 40 00:02:10,500 --> 00:02:12,030 define chemical reactivity-- 41 00:02:12,030 --> 00:02:14,760 I think that's a key issue-- 42 00:02:14,760 --> 00:02:18,300 and then what are defense mechanisms 43 00:02:18,300 --> 00:02:21,030 against these reactive oxygen species, 44 00:02:21,030 --> 00:02:27,870 before then focusing on the NADPH oxidases, which those are 45 00:02:27,870 --> 00:02:31,110 the enzymes that we're focused on in this whole module 46 00:02:31,110 --> 00:02:34,440 on reactive oxygen species. 47 00:02:34,440 --> 00:02:41,640 So identification, and I had put this 48 00:02:41,640 --> 00:02:43,770 on the board the last time. 49 00:02:43,770 --> 00:02:47,310 We have-- just repeating what's over there, 50 00:02:47,310 --> 00:02:56,630 superoxide hydroxide radical, hydrogen peroxide. 51 00:02:56,630 --> 00:03:01,607 Because this is together, and hypochlorous acid. 52 00:03:01,607 --> 00:03:03,940 And we're going to look at the reactivity of these guys. 53 00:03:03,940 --> 00:03:06,580 These are one electron oxidants. 54 00:03:06,580 --> 00:03:08,910 These are two electron oxidants. 55 00:03:08,910 --> 00:03:13,200 So reactive species don't need to have free radicals. 56 00:03:13,200 --> 00:03:15,750 They can do two electron chemistry, 57 00:03:15,750 --> 00:03:17,960 or they can do one electron chemistry. 58 00:03:17,960 --> 00:03:22,040 OK, so a key thing to think about, 59 00:03:22,040 --> 00:03:25,940 and I don't expect you to remember the detailed reduction 60 00:03:25,940 --> 00:03:29,750 potentials, but again, as the inadvertent consequence 61 00:03:29,750 --> 00:03:34,910 of our environment, you know, 1.8 to 0.8 billion years ago we 62 00:03:34,910 --> 00:03:37,040 moved from an anaerobic to an aerobic world, 63 00:03:37,040 --> 00:03:39,090 where we have metals all over the place. 64 00:03:39,090 --> 00:03:43,200 The question is how do you control these reactivities. 65 00:03:43,200 --> 00:03:47,000 And so we need to think about the redox chemistry of oxygen. 66 00:03:47,000 --> 00:03:55,640 So oxygen, and the details the actual reduction potentials, 67 00:03:55,640 --> 00:03:58,190 and these are some of them given here. 68 00:03:58,190 --> 00:04:01,880 And this one somehow got lost from here 69 00:04:01,880 --> 00:04:03,710 to here, which is 0.94. 70 00:04:03,710 --> 00:04:07,970 But it's in a table later on in the PowerPoints, 71 00:04:07,970 --> 00:04:10,667 depend on the balanced equation. 72 00:04:10,667 --> 00:04:12,500 So if you're looking at this, and you really 73 00:04:12,500 --> 00:04:14,600 want to think about the reduction potentials, 74 00:04:14,600 --> 00:04:17,600 you need to count the numbers of electrons and protons 75 00:04:17,600 --> 00:04:19,610 and balance the equation that you're 76 00:04:19,610 --> 00:04:23,220 looking at, because the reduction potentials vary. 77 00:04:23,220 --> 00:04:28,070 But what you need to remember is the more positive 78 00:04:28,070 --> 00:04:33,950 the number is, the easier it is to reduce, the more powerful 79 00:04:33,950 --> 00:04:35,270 the oxidant. 80 00:04:35,270 --> 00:04:39,650 So we're going to have oxygen. And in the first one, 81 00:04:39,650 --> 00:04:42,260 we have one electron reduction. 82 00:04:42,260 --> 00:04:47,780 And this is the only one where the reduction potential 83 00:04:47,780 --> 00:04:49,040 is negative. 84 00:04:49,040 --> 00:04:50,360 So this is uphill. 85 00:04:50,360 --> 00:04:52,820 It doesn't want to be oxidized. 86 00:04:52,820 --> 00:05:07,460 And this produces superoxide So this is one of the guys 87 00:05:07,460 --> 00:05:10,010 we're going to be talking about, and here's 88 00:05:10,010 --> 00:05:11,630 the reduction potential. 89 00:05:11,630 --> 00:05:15,800 This can be further reduced with an electron 90 00:05:15,800 --> 00:05:21,812 and a couple of protons to form hydrogen peroxide. 91 00:05:21,812 --> 00:05:23,270 So I'm not going to write that out. 92 00:05:23,270 --> 00:05:26,770 Everybody hopefully knows what hydrogen peroxide is, 93 00:05:26,770 --> 00:05:29,530 and this is the number that for some reason 94 00:05:29,530 --> 00:05:34,340 got left off that handout, and this is favorable. 95 00:05:37,190 --> 00:05:39,730 And so are all the other numbers. 96 00:05:39,730 --> 00:05:42,280 And the numbers are actually large and favorable. 97 00:05:42,280 --> 00:05:44,590 That means they're good oxidants. 98 00:05:44,590 --> 00:05:53,455 OK, so more positive, better oxidants. 99 00:05:56,860 --> 00:05:59,530 These are all biological reduction potentials, 100 00:05:59,530 --> 00:06:02,380 relative to the normal hydrogen electrode. 101 00:06:02,380 --> 00:06:05,470 So these are the ones-- there's actually a table in there 102 00:06:05,470 --> 00:06:08,860 where you see the numbers that people 103 00:06:08,860 --> 00:06:14,150 interested in biological systems focus on. 104 00:06:14,150 --> 00:06:22,400 Now, we'll see later on that hydrogen peroxide gives rise 105 00:06:22,400 --> 00:06:25,180 to hypochlorous acid in the presence of chloride. 106 00:06:27,950 --> 00:06:36,400 But hydrogen peroxide can also get further reduced to-- 107 00:06:36,400 --> 00:06:39,080 and this is one where I don't have the balanced equation 108 00:06:39,080 --> 00:06:41,610 to hydroxide radical. 109 00:06:41,610 --> 00:06:46,610 And so the number I have written down here is 0.38 volts. 110 00:06:46,610 --> 00:06:50,540 But again, the numbers aren't so important. 111 00:06:50,540 --> 00:06:55,760 And then this can get further reduced to water, 112 00:06:55,760 --> 00:06:58,910 and this number is-- 113 00:06:58,910 --> 00:07:00,620 you'll see in a number of the tables 114 00:07:00,620 --> 00:07:04,370 I give you is 1.31 volts. 115 00:07:04,370 --> 00:07:07,670 So we have different states, and all three states, 116 00:07:07,670 --> 00:07:09,770 consequence of moving from the anaerobic 117 00:07:09,770 --> 00:07:12,410 to the aerobic world that give you species that are 118 00:07:12,410 --> 00:07:15,230 called reactive oxygen species. 119 00:07:15,230 --> 00:07:19,670 And the one that's hardest to form is superoxide 120 00:07:19,670 --> 00:07:23,420 and this is the one that's probably the least reactive. 121 00:07:23,420 --> 00:07:28,610 But what I'm going to do is give you a couple sets of criteria 122 00:07:28,610 --> 00:07:31,460 for reactivity, because when people say something 123 00:07:31,460 --> 00:07:36,230 is chemically reactive, you know, what does that mean? 124 00:07:36,230 --> 00:07:39,440 It depends on what it is reacting with. 125 00:07:39,440 --> 00:07:42,230 And so this, I think, is the real problem 126 00:07:42,230 --> 00:07:44,030 in the field is that people really 127 00:07:44,030 --> 00:07:47,010 don't define this very well. 128 00:07:47,010 --> 00:07:48,960 And so this is why I think the Winterbourn 129 00:07:48,960 --> 00:07:51,290 paper is so important. 130 00:07:51,290 --> 00:07:52,520 So she has a table in there. 131 00:07:52,520 --> 00:07:54,062 I'm going to have the table up there. 132 00:07:54,062 --> 00:07:57,290 I reorganized the table to focus on what 133 00:07:57,290 --> 00:08:02,300 I want to focus in this brief introduction to this topic 134 00:08:02,300 --> 00:08:04,700 and show you how they define reactivity. 135 00:08:04,700 --> 00:08:06,980 But if you are thinking about something, 136 00:08:06,980 --> 00:08:10,190 you need to define what the reaction is 137 00:08:10,190 --> 00:08:15,530 that you're interested in with any of these reactive oxygen 138 00:08:15,530 --> 00:08:18,410 species. 139 00:08:18,410 --> 00:08:22,080 So these are the guys we care about. 140 00:08:22,080 --> 00:08:27,080 And the second question I wanted to address is-- 141 00:08:27,080 --> 00:08:32,210 so first we were identifying last time the identification. 142 00:08:32,210 --> 00:08:34,190 Second is a chemical reactivity. 143 00:08:40,900 --> 00:08:42,030 OK. 144 00:08:42,030 --> 00:08:45,510 And I've taken this-- 145 00:08:45,510 --> 00:08:46,830 maybe it wasn't from her paper. 146 00:08:46,830 --> 00:08:48,180 I can't remember where I've taken this. 147 00:08:48,180 --> 00:08:49,347 I should have referenced it. 148 00:08:49,347 --> 00:08:53,100 But anyhow, what I'm going to do is give you a simple table 149 00:08:53,100 --> 00:08:56,070 that I think is useful to think about reactivity. 150 00:08:56,070 --> 00:08:58,232 So if we look at reactivity. 151 00:08:58,232 --> 00:08:59,690 We're going to look at the oxidant. 152 00:09:03,540 --> 00:09:05,950 And then another category is going 153 00:09:05,950 --> 00:09:08,790 to be the biological defense. 154 00:09:11,550 --> 00:09:18,960 And the third category is thermodynamic properties. 155 00:09:18,960 --> 00:09:22,140 And you all know that things can be thermodynamically favorable, 156 00:09:22,140 --> 00:09:25,740 but not happen at all, like oxygen oxidizing glucose 157 00:09:25,740 --> 00:09:27,510 on the table. 158 00:09:27,510 --> 00:09:30,360 So you not only need to think about the thermodynamics, which 159 00:09:30,360 --> 00:09:33,000 involves the redox potentials, and you 160 00:09:33,000 --> 00:09:34,975 need to define the sorts of conditions 161 00:09:34,975 --> 00:09:36,600 that you're looking under, but then you 162 00:09:36,600 --> 00:09:38,470 need to also think about the kinetics. 163 00:09:38,470 --> 00:09:40,770 OK, so we have thermodynamics. 164 00:09:40,770 --> 00:09:46,260 So we have biological defense, thermodynamics, and then 165 00:09:46,260 --> 00:09:47,490 the kinetic properties. 166 00:09:50,130 --> 00:09:54,240 And the kinetic properties are often 167 00:09:54,240 --> 00:10:01,260 given in terms of reactivity with a molecule 168 00:10:01,260 --> 00:10:02,640 called glutathione. 169 00:10:05,433 --> 00:10:07,100 I'm not going to draw out the structure. 170 00:10:07,100 --> 00:10:11,980 But it's a tripeptide with gamma glutamyl cystine glycine. 171 00:10:11,980 --> 00:10:16,790 OK, so it's a tripeptide with an unusual linkage 172 00:10:16,790 --> 00:10:19,130 to the next amino acid. 173 00:10:19,130 --> 00:10:20,630 What do you know about glutathione? 174 00:10:20,630 --> 00:10:23,180 Have you guys ever seen that before? 175 00:10:23,180 --> 00:10:27,440 So it's a major redox buffer inside human cells. 176 00:10:27,440 --> 00:10:29,480 Now, if you're in a microorganism, 177 00:10:29,480 --> 00:10:32,330 we don't use the same major redox, 178 00:10:32,330 --> 00:10:33,620 so you need to look at that. 179 00:10:33,620 --> 00:10:36,530 But all organisms have redox buffers. 180 00:10:36,530 --> 00:10:39,020 And as you can imagine, and this is 181 00:10:39,020 --> 00:10:43,670 why we focused with the mass spec stuff, sulfenylation, 182 00:10:43,670 --> 00:10:48,710 one of the major targets of out of control reactive oxygen 183 00:10:48,710 --> 00:10:51,620 species is oxidation of cystines. 184 00:10:51,620 --> 00:10:53,840 There are other amino acids that get oxidized, 185 00:10:53,840 --> 00:10:56,540 but the focus is on the cystine [INAUDIBLE] 186 00:10:56,540 --> 00:10:58,190 and all the changes that can be made 187 00:10:58,190 --> 00:11:01,190 and all the signaling, most of the signaling, 188 00:11:01,190 --> 00:11:04,340 through reactive oxygen species all 189 00:11:04,340 --> 00:11:06,770 go through cystine oxidation. 190 00:11:06,770 --> 00:11:10,890 So this is a reasonable choice, gamma glutamate. 191 00:11:10,890 --> 00:11:13,610 So let me just write down, it's a tripeptide 192 00:11:13,610 --> 00:11:18,410 of glutamate cystine and glycine, 193 00:11:18,410 --> 00:11:24,110 with an unusual linkage here, with an iso peptide linkage. 194 00:11:24,110 --> 00:11:31,180 So the first two are one electron oxidants, 195 00:11:31,180 --> 00:11:35,240 and the first one to talk about is hydroxide radical. 196 00:11:35,240 --> 00:11:37,640 Hydroxide radical you'll see by far and away 197 00:11:37,640 --> 00:11:42,350 is one of the most reactive, is dying to be reduced, 198 00:11:42,350 --> 00:11:45,500 as you can tell, by this reduction potential, 199 00:11:45,500 --> 00:11:48,770 no matter what the variation on the theme is. 200 00:11:48,770 --> 00:11:51,350 And there is no defense. 201 00:11:51,350 --> 00:11:54,230 So you don't want to get to hydroxide radical. 202 00:11:54,230 --> 00:12:00,780 So there's no actual defense. 203 00:12:00,780 --> 00:12:04,780 And that's not completely true, because in reality, 204 00:12:04,780 --> 00:12:08,760 if you have glutathione around, the glutathione will reduce 205 00:12:08,760 --> 00:12:11,270 this by hydrogen atom transfer. 206 00:12:11,270 --> 00:12:15,920 So an important component is the redox buffer. 207 00:12:15,920 --> 00:12:18,297 So glutathione-- let me just write that down again, 208 00:12:18,297 --> 00:12:20,630 because we're not going to have time to talk about this, 209 00:12:20,630 --> 00:12:30,140 but redox buffers play a central role in reactive oxygen 210 00:12:30,140 --> 00:12:32,870 species. 211 00:12:32,870 --> 00:12:39,260 And so the thermodynamics of this, it's dying to be reduced. 212 00:12:39,260 --> 00:12:41,000 If I have the numbers right, I think-- 213 00:12:41,000 --> 00:12:42,625 I don't remember what the numbers were. 214 00:12:42,625 --> 00:12:45,010 OK, so the numbers here are 0.31. 215 00:12:45,010 --> 00:12:48,240 I have different numbers in different places. 216 00:12:48,240 --> 00:12:49,490 But anyhow, it doesn't matter. 217 00:12:49,490 --> 00:12:51,440 It's dying to be reduced. 218 00:12:51,440 --> 00:12:54,530 It's a hot oxidant. 219 00:12:54,530 --> 00:12:59,810 And then the rate constant for reaction with glutathione. 220 00:12:59,810 --> 00:13:03,170 OK, so it would be h dot transfer. 221 00:13:03,170 --> 00:13:07,190 And this is a bimolecular. 222 00:13:07,190 --> 00:13:16,590 Rate constant is 1 times 10 to the 10th per molar per second. 223 00:13:16,590 --> 00:13:18,960 So this is really fast. 224 00:13:18,960 --> 00:13:21,440 And so if you've got glutathione around, 225 00:13:21,440 --> 00:13:23,060 your hydroxide radical is gone. 226 00:13:23,060 --> 00:13:25,280 We're going to look at another way of trying 227 00:13:25,280 --> 00:13:28,160 to define reactivity, but this is the way 228 00:13:28,160 --> 00:13:31,160 that people, who were trying to think about the kinetics of all 229 00:13:31,160 --> 00:13:33,620 this, are starting to do this. 230 00:13:33,620 --> 00:13:37,160 OK, so this is one. 231 00:13:37,160 --> 00:13:42,560 The second species, which is also a one electron reductant, 232 00:13:42,560 --> 00:13:46,480 is superoxide And this is the one 233 00:13:46,480 --> 00:13:50,930 seen described most frequently as a reactive oxygen species. 234 00:13:50,930 --> 00:13:54,170 In reality, it's not very reactive at all. 235 00:13:54,170 --> 00:13:57,020 It is reactive, but not anywhere near 236 00:13:57,020 --> 00:14:01,130 as reactive as some of the others. 237 00:14:01,130 --> 00:14:03,295 And do we have a defense mechanism? 238 00:14:03,295 --> 00:14:04,670 We'll come back to this a little, 239 00:14:04,670 --> 00:14:06,128 and I'll write a balanced equation. 240 00:14:06,128 --> 00:14:07,670 I'm just going to list things. 241 00:14:07,670 --> 00:14:12,440 We have enzymes, called SODs, and so this 242 00:14:12,440 --> 00:14:17,240 is superoxide dismutase. 243 00:14:17,240 --> 00:14:20,690 And I'll come back and write a balanced equation in a minute. 244 00:14:20,690 --> 00:14:24,620 So we have proteins that are devoted to this, but in reality 245 00:14:24,620 --> 00:14:28,160 metals, like manganese inside the cell 246 00:14:28,160 --> 00:14:32,750 can actually function as a superoxide dismutase 247 00:14:32,750 --> 00:14:34,610 at reasonable rates. 248 00:14:34,610 --> 00:14:41,630 Protons cause rapid dismutation to form hydrogen peroxide 249 00:14:41,630 --> 00:14:43,290 and oxygen. 250 00:14:43,290 --> 00:14:45,760 This guy is also dying to be reduced, 251 00:14:45,760 --> 00:14:49,850 so thermodynamically, this is a good oxidant. 252 00:14:49,850 --> 00:14:53,000 But the key is thinking about the kinetics. 253 00:14:53,000 --> 00:14:56,780 And it obviously depends on the reaction you're looking at, 254 00:14:56,780 --> 00:14:58,700 the kinetics are going to be different 255 00:14:58,700 --> 00:15:03,320 with every small molecule or large molecule it interacts 256 00:15:03,320 --> 00:15:03,820 with. 257 00:15:03,820 --> 00:15:06,950 But, again, we're using glutathione as an example. 258 00:15:06,950 --> 00:15:11,870 And the numbers that people report for superoxide compared 259 00:15:11,870 --> 00:15:21,080 to 1 times tend to the 10th are now 10 to 1,000 260 00:15:21,080 --> 00:15:24,150 per molar per second. 261 00:15:24,150 --> 00:15:28,190 OK, so this is chemically much less reactive than hydroxide 262 00:15:28,190 --> 00:15:29,750 radical. 263 00:15:29,750 --> 00:15:34,560 And even for this one, we have a defense mechanism. 264 00:15:34,560 --> 00:15:37,040 This one, again, is problematic. 265 00:15:37,040 --> 00:15:39,560 OK, so now what we're going to switch to 266 00:15:39,560 --> 00:15:45,410 is two electron oxidants. 267 00:15:45,410 --> 00:15:50,360 And the one we're going to focus on today, 268 00:15:50,360 --> 00:15:54,260 in this section, what happens in neutrophils 269 00:15:54,260 --> 00:15:57,920 to defend against invading organisms, like bacteria 270 00:15:57,920 --> 00:16:01,100 or viruses or parasites. 271 00:16:01,100 --> 00:16:06,530 The major way that this becomes neutralized inside the cell 272 00:16:06,530 --> 00:16:09,590 is, again, in a mammalian cell is with glutathione. 273 00:16:09,590 --> 00:16:11,180 So this is a small molecule. 274 00:16:16,290 --> 00:16:19,110 It also is a very strong oxidant, 275 00:16:19,110 --> 00:16:25,290 but the mechanism of oxidation is distinct, two electrons, 276 00:16:25,290 --> 00:16:26,370 versus one electron. 277 00:16:26,370 --> 00:16:29,820 We're going to look at examples of this. 278 00:16:29,820 --> 00:16:32,640 And if you look at the rate constant for reaction 279 00:16:32,640 --> 00:16:34,890 with glutathione-- 280 00:16:34,890 --> 00:16:37,650 And again, you need to really think 281 00:16:37,650 --> 00:16:42,390 about a balanced equation in the kinetics of all 282 00:16:42,390 --> 00:16:43,310 of these things. 283 00:16:43,310 --> 00:16:45,060 If you're ever going to work in this area, 284 00:16:45,060 --> 00:16:46,185 that's what you need to do. 285 00:16:46,185 --> 00:16:48,300 You need to educate yourself about what 286 00:16:48,300 --> 00:16:51,480 the species are with which you're going to interact. 287 00:16:51,480 --> 00:16:55,650 But you can see from this number under the sets of conditions, 288 00:16:55,650 --> 00:16:57,540 they did everything the same way, 289 00:16:57,540 --> 00:17:01,140 so that they could compare the relative reactivity 290 00:17:01,140 --> 00:17:03,690 of these molecules, 2 times 10 to the 7th. 291 00:17:03,690 --> 00:17:08,160 So this is much more reactive, for example, in superoxide 292 00:17:08,160 --> 00:17:17,750 And then the last one is hydrogen peroxide. 293 00:17:17,750 --> 00:17:19,300 So this is also two electron. 294 00:17:24,349 --> 00:17:27,109 And two electron, we will see that there 295 00:17:27,109 --> 00:17:34,068 are a number of proteins that mount a defense. 296 00:17:34,068 --> 00:17:36,110 These are called-- and I'm going to show you this 297 00:17:36,110 --> 00:17:37,670 in a minute-- paroxyredoxans. 298 00:17:41,210 --> 00:17:43,970 I'll show you what they do. 299 00:17:43,970 --> 00:17:45,920 What did you see-- do you remember 300 00:17:45,920 --> 00:17:51,720 the enzyme that was used in the Carroll paper this week 301 00:17:51,720 --> 00:17:53,090 in recitation? 302 00:17:53,090 --> 00:17:55,250 To get rid of hydrogen peroxide, what did they use? 303 00:17:55,250 --> 00:17:56,060 Anybody remember? 304 00:18:01,380 --> 00:18:02,660 We use catalase. 305 00:18:02,660 --> 00:18:07,320 I'm going to come back and write the equation, so catalase, 306 00:18:07,320 --> 00:18:10,440 and then the other one, which we also talked about, 307 00:18:10,440 --> 00:18:13,890 but we didn't talk about the chemistry in the Carroll paper, 308 00:18:13,890 --> 00:18:16,030 was peroxyredoxins. 309 00:18:16,030 --> 00:18:18,280 And there were like seven or eight different isozymes. 310 00:18:18,280 --> 00:18:23,910 So we have a number of ways of dealing with hydrogen peroxide. 311 00:18:23,910 --> 00:18:32,490 Again, it is thermodynamically favorable to be an oxidant. 312 00:18:32,490 --> 00:18:34,650 But as we've already talked about before, 313 00:18:34,650 --> 00:18:37,800 hydrogen peroxide is really not very chemically reactive 314 00:18:37,800 --> 00:18:38,790 at all. 315 00:18:38,790 --> 00:18:44,900 And so the numbers that they quote 316 00:18:44,900 --> 00:18:50,130 under these sets of conditions are 0.9 per molar per second. 317 00:18:50,130 --> 00:18:52,340 So it's much, much slower. 318 00:18:52,340 --> 00:18:58,640 You see numbers that range from 0.9 to 20, 319 00:18:58,640 --> 00:19:01,670 but this has really important implications 320 00:19:01,670 --> 00:19:05,390 in the paper we talked about in the mass spec analysis, where 321 00:19:05,390 --> 00:19:08,990 hydrogen peroxide is functioning as a signaling agent. 322 00:19:08,990 --> 00:19:11,390 We're going to come back to this later on. 323 00:19:11,390 --> 00:19:15,260 And this for years made more chemical-y type people not 324 00:19:15,260 --> 00:19:18,410 believe that hydrogen peroxide was involved in signaling, 325 00:19:18,410 --> 00:19:19,910 because the rate constants were just 326 00:19:19,910 --> 00:19:22,730 too slow, compared to the biological response 327 00:19:22,730 --> 00:19:25,040 of the other side. 328 00:19:25,040 --> 00:19:32,390 So this is sort of a superficial overview of the differences 329 00:19:32,390 --> 00:19:34,910 in reactivities, but the real take home message 330 00:19:34,910 --> 00:19:37,640 is these molecules have different chemistry 331 00:19:37,640 --> 00:19:39,380 and different reactivities. 332 00:19:39,380 --> 00:19:42,877 And I guarantee if you're studying stuff inside the cell, 333 00:19:42,877 --> 00:19:44,960 you're going to worry about these kinds of things, 334 00:19:44,960 --> 00:19:48,260 and you need to educate yourself about what you're 335 00:19:48,260 --> 00:19:50,900 worried about in terms of the redox 336 00:19:50,900 --> 00:19:53,720 potentials of these systems. 337 00:19:53,720 --> 00:19:55,325 So there's a second way. 338 00:19:57,990 --> 00:20:00,390 So those are just the redox potentials. 339 00:20:00,390 --> 00:20:04,770 So there's a second way to look at reactivity, 340 00:20:04,770 --> 00:20:07,710 and this is also, I think, in the paper you had to read. 341 00:20:07,710 --> 00:20:20,020 So the second way is by just looking at diffusion, how far-- 342 00:20:20,020 --> 00:20:21,110 this is within a cell-- 343 00:20:26,150 --> 00:20:29,530 can you still feel the effects of the oxidant. 344 00:20:39,880 --> 00:20:43,330 And so I'm going to say see PowerPoint for the cartoon. 345 00:20:46,240 --> 00:20:49,840 So I think this is a good way to look at this. 346 00:20:49,840 --> 00:20:54,040 And again, the numbers are squishy, 347 00:20:54,040 --> 00:20:56,650 but here we are inside-- 348 00:20:56,650 --> 00:20:58,660 this is the cell, and the question 349 00:20:58,660 --> 00:21:01,690 is, do some of these oxidants get out of the cell 350 00:21:01,690 --> 00:21:04,250 and go to the next sets of cells. 351 00:21:04,250 --> 00:21:08,870 And so if you look at something like hydrogen peroxide. 352 00:21:08,870 --> 00:21:12,730 So hydrogen peroxide is the least reactive 353 00:21:12,730 --> 00:21:15,100 from this criteria of kinetically, the least 354 00:21:15,100 --> 00:21:15,850 reactive. 355 00:21:15,850 --> 00:21:18,970 And it goes way outside the cell. 356 00:21:18,970 --> 00:21:22,160 So it diffuses farthest away. 357 00:21:22,160 --> 00:21:25,670 So that means it's the least reactive. 358 00:21:25,670 --> 00:21:33,840 So the distance is used to define reactivity. 359 00:21:33,840 --> 00:21:35,440 And again, this is a squishy number, 360 00:21:35,440 --> 00:21:36,730 but I think it's informative. 361 00:21:36,730 --> 00:21:40,180 Now, what you see here-- so hydrogen peroxide, we just 362 00:21:40,180 --> 00:21:42,700 went through, is the least reactive. 363 00:21:42,700 --> 00:21:46,570 But I also told you that hydrogen peroxide, 364 00:21:46,570 --> 00:21:50,800 there are many ways to remove it inside the cell, 365 00:21:50,800 --> 00:21:54,370 peroxyredoxins, glutathione, glutathione peroxidases 366 00:21:54,370 --> 00:21:56,740 catalases. 367 00:21:56,740 --> 00:21:58,210 They all remove it. 368 00:21:58,210 --> 00:22:00,490 They all have different rate constants. 369 00:22:00,490 --> 00:22:03,250 Peroxidative redoxins account for, I think, 370 00:22:03,250 --> 00:22:09,610 it's 1.5% of mammalian cells, and they're 371 00:22:09,610 --> 00:22:12,970 very important in controlling redox balance. 372 00:22:12,970 --> 00:22:15,250 And what do you see here? 373 00:22:15,250 --> 00:22:19,620 If you are in an environment where you have a peroxyredoxin, 374 00:22:19,620 --> 00:22:20,560 what happens? 375 00:22:20,560 --> 00:22:23,800 This diffuses a lot less quickly. 376 00:22:23,800 --> 00:22:24,790 Why? 377 00:22:24,790 --> 00:22:28,480 Because the enzyme rapidly reacts with this molecule. 378 00:22:28,480 --> 00:22:31,510 So we know that the enzyme can react. 379 00:22:31,510 --> 00:22:33,190 I haven't given you that number. 380 00:22:33,190 --> 00:22:36,580 But we'll see that this number is on-- instead 381 00:22:36,580 --> 00:22:40,210 of being a number of 0.9 to 20, is 382 00:22:40,210 --> 00:22:45,710 going to be 10 to the 6th per molar per second. 383 00:22:45,710 --> 00:22:49,480 So this now-- something about the active site 384 00:22:49,480 --> 00:22:52,960 of this-- and it's not SH versus thiolate. 385 00:22:52,960 --> 00:22:54,000 We all learn now. 386 00:22:54,000 --> 00:22:55,270 Everybody is good at this. 387 00:22:55,270 --> 00:22:57,040 Thiolates are the reactive species. 388 00:22:57,040 --> 00:22:58,550 It has nothing to do with that. 389 00:22:58,550 --> 00:23:02,170 Thiolates are always more reactive. 390 00:23:02,170 --> 00:23:03,880 But there's something else special 391 00:23:03,880 --> 00:23:07,180 about these proteins that allow them 392 00:23:07,180 --> 00:23:09,310 to control hydrogen peroxide. 393 00:23:09,310 --> 00:23:11,620 Now why might you want to do this? 394 00:23:11,620 --> 00:23:15,220 If you have a signaling agent, like hydrogen peroxide, 395 00:23:15,220 --> 00:23:17,950 you don't want it going all the way over here. 396 00:23:17,950 --> 00:23:23,230 You want to control the effective concentration near we 397 00:23:23,230 --> 00:23:25,400 want the chemistry to happen. 398 00:23:25,400 --> 00:23:28,990 So these peroxyredoxins play an incredibly important role 399 00:23:28,990 --> 00:23:32,300 in controlling the effective concentrations. 400 00:23:32,300 --> 00:23:35,140 And so if you look here within the cell, again, 401 00:23:35,140 --> 00:23:37,570 we're only focusing on oxygen, but 402 00:23:37,570 --> 00:23:43,750 both hydroxide radical and hypochlorous acid 403 00:23:43,750 --> 00:23:45,670 are very, very reactive. 404 00:23:45,670 --> 00:23:48,730 You can't go very far without having 405 00:23:48,730 --> 00:23:51,550 them react with something, and that, again, 406 00:23:51,550 --> 00:23:55,450 is consistent with the kinetic analyses 407 00:23:55,450 --> 00:23:59,530 that people have done over the years. 408 00:23:59,530 --> 00:24:02,980 So the take home message from all of that 409 00:24:02,980 --> 00:24:07,510 is that these reactive oxygen species have 410 00:24:07,510 --> 00:24:09,510 different chemistry and different reactivities, 411 00:24:09,510 --> 00:24:10,968 and you've got to educate yourself. 412 00:24:10,968 --> 00:24:14,730 But some of these things, HOCL and hydroxide radical, 413 00:24:14,730 --> 00:24:17,710 are very reactive, no matter what. 414 00:24:17,710 --> 00:24:19,900 So the last thing I wanted to focus on 415 00:24:19,900 --> 00:24:25,990 was in this section, which is basically the introduction, 416 00:24:25,990 --> 00:24:28,610 is the defense mechanisms. 417 00:24:28,610 --> 00:24:30,810 OK, so this is the defense. 418 00:24:30,810 --> 00:24:36,490 And I already have listed what the defense mechanisms are, 419 00:24:36,490 --> 00:24:39,015 but I wanted to give you a few equations. 420 00:24:39,015 --> 00:24:40,390 You've already seen that they can 421 00:24:40,390 --> 00:24:47,020 be enzymes or small molecules. 422 00:24:51,350 --> 00:24:58,870 And so one example we already looked at is-- 423 00:24:58,870 --> 00:25:02,850 we already described, but didn't look at in chemical details 424 00:25:02,850 --> 00:25:04,740 is superoxide dismutase. 425 00:25:04,740 --> 00:25:08,950 OK, what does superoxide dismutase do? 426 00:25:08,950 --> 00:25:14,020 It takes two molecules of superoxide 427 00:25:14,020 --> 00:25:19,600 and they disproportionate in the presence of protons 428 00:25:19,600 --> 00:25:27,220 to hydrogen peroxide and oxygen. And these enzymes 429 00:25:27,220 --> 00:25:31,480 have a kcat over km, a catalytic efficiency 430 00:25:31,480 --> 00:25:38,650 on the order of 7 times 10 to the 9th per molar per second. 431 00:25:38,650 --> 00:25:41,080 So these are incredibly efficient. 432 00:25:41,080 --> 00:25:44,420 In fact, metals-- again, manganese in solution, 433 00:25:44,420 --> 00:25:46,420 in some organisms, they have a lot of manganese, 434 00:25:46,420 --> 00:25:48,490 they can actually do disproportionation. 435 00:25:48,490 --> 00:25:50,920 But it's all about the rate constants. 436 00:25:50,920 --> 00:25:53,020 So this is incredibly efficient. 437 00:25:53,020 --> 00:25:56,430 These enzymes are in all organisms. 438 00:25:56,430 --> 00:26:00,730 And obviously, this reaction is very important. 439 00:26:00,730 --> 00:26:05,500 You don't want superoxide completely uncontrolled, 440 00:26:05,500 --> 00:26:08,390 and there are some of these enzymes-- these are 441 00:26:08,390 --> 00:26:10,570 all metal catalyzed reactions. 442 00:26:10,570 --> 00:26:12,310 Some use iron. 443 00:26:12,310 --> 00:26:14,440 Some use manganese. 444 00:26:14,440 --> 00:26:16,420 Some use copper. 445 00:26:16,420 --> 00:26:19,780 Some use-- humans use copper and zinc. 446 00:26:19,780 --> 00:26:22,990 And there are others that use nickel. 447 00:26:22,990 --> 00:26:24,790 And they all have different properties, 448 00:26:24,790 --> 00:26:27,290 and they've all been studied in some fashion. 449 00:26:27,290 --> 00:26:29,950 So depending on where the organism lives, 450 00:26:29,950 --> 00:26:33,880 they might use different superoxide dismutases 451 00:26:33,880 --> 00:26:38,950 to control the levels of this reoxygen species. 452 00:26:38,950 --> 00:26:41,440 The second defense mixing mechanism 453 00:26:41,440 --> 00:26:44,080 of the peroxyredoxins. 454 00:26:44,080 --> 00:26:48,890 I think I have this one up here. 455 00:26:48,890 --> 00:26:52,910 So any of you that are interested in this, there 456 00:26:52,910 --> 00:26:54,830 were like a seven or eight isozymes. 457 00:26:54,830 --> 00:26:58,710 They keep finding new isozymes everywhere inside the cell. 458 00:26:58,710 --> 00:27:00,160 They are at high concentrations. 459 00:27:00,160 --> 00:27:03,410 They are clearly very important in controlling the redox 460 00:27:03,410 --> 00:27:04,580 balance. 461 00:27:04,580 --> 00:27:09,650 So they do react with hydrogen peroxide, 462 00:27:09,650 --> 00:27:13,280 but they also react with other peroxides, 463 00:27:13,280 --> 00:27:17,240 and they're important in controlling the redox balance. 464 00:27:21,340 --> 00:27:25,070 And so, here and in each one of these 465 00:27:25,070 --> 00:27:29,030 isozymes has its own characteristics. 466 00:27:29,030 --> 00:27:30,900 You don't need to remember the details, 467 00:27:30,900 --> 00:27:33,560 but the chemical mechanisms of sort of the same, 468 00:27:33,560 --> 00:27:38,300 even though some are dimers, some are monomers. 469 00:27:38,300 --> 00:27:42,410 It turns out they all have in the monomer two 470 00:27:42,410 --> 00:27:45,200 reactive cystines. 471 00:27:45,200 --> 00:27:47,900 So one is called Cp. 472 00:27:47,900 --> 00:27:51,950 That means that's the species that reacts with the hydrogen 473 00:27:51,950 --> 00:27:53,400 peroxide. 474 00:27:53,400 --> 00:27:58,160 So we have-- they can be monomers or dimers. 475 00:27:58,160 --> 00:28:01,370 This is the protein. 476 00:28:01,370 --> 00:28:09,330 And so you could have a Cp which can react to get sulfenylated. 477 00:28:14,970 --> 00:28:21,120 And then you have CR, which can react to resolve 478 00:28:21,120 --> 00:28:22,980 the sulfenylation process. 479 00:28:22,980 --> 00:28:25,800 So you're going to get rid of the sulfenic acid. 480 00:28:25,800 --> 00:28:32,132 So here, if you have a CR I'm being sloppy here. 481 00:28:32,132 --> 00:28:33,590 In other words, you probably have-- 482 00:28:33,590 --> 00:28:34,820 this is probably protinated. 483 00:28:34,820 --> 00:28:39,660 It's all controlled to generate the anionic form of the file, 484 00:28:39,660 --> 00:28:41,780 which then can form-- 485 00:28:41,780 --> 00:28:45,630 in this case, I'm drawing an intramolecular disulfide. 486 00:28:45,630 --> 00:28:48,240 OK, so this forms a disulfide. 487 00:28:51,680 --> 00:28:54,610 And this is intramolecular. 488 00:28:58,460 --> 00:29:02,480 So what do we see over here? 489 00:29:02,480 --> 00:29:07,250 Over here, we see you can form an intermolecular disulfide, 490 00:29:07,250 --> 00:29:10,250 if the molecule's a dimer. 491 00:29:10,250 --> 00:29:12,680 So the chemistry is exactly the same. 492 00:29:12,680 --> 00:29:14,630 But sometimes that occurs to the monomer. 493 00:29:14,630 --> 00:29:18,170 Sometimes it occurs through the dimer. 494 00:29:18,170 --> 00:29:20,810 And then the question is once you have the disulfide, 495 00:29:20,810 --> 00:29:21,840 so now you have-- 496 00:29:25,760 --> 00:29:29,210 how do you re-reduce this? 497 00:29:29,210 --> 00:29:33,500 And you re-reduce this by some kind of reductant, 498 00:29:33,500 --> 00:29:35,240 such as thioredoxin which we will 499 00:29:35,240 --> 00:29:37,670 see if we get it as far as talking 500 00:29:37,670 --> 00:29:40,250 about ribonucleotide reductase. 501 00:29:40,250 --> 00:29:46,820 So where have you seen these kinds of proteins before? 502 00:29:46,820 --> 00:29:48,890 Does anybody remember. 503 00:29:48,890 --> 00:29:54,650 So thioredoxin-- this is thioredoxin. 504 00:29:54,650 --> 00:29:58,670 There are probably 10 different kinds 505 00:29:58,670 --> 00:30:01,880 of thioredoxins inside the cell, these small little proteins, 506 00:30:01,880 --> 00:30:04,400 as is peroxiredoxin. 507 00:30:04,400 --> 00:30:07,750 And they're all involved really in redox balance. 508 00:30:07,750 --> 00:30:11,000 So we can intercept the hydrogen peroxide. 509 00:30:11,000 --> 00:30:13,885 Say we want to get rid of the hydrogen peroxide fast, 510 00:30:13,885 --> 00:30:16,010 we've done our signaling, we want to get rid of it, 511 00:30:16,010 --> 00:30:17,720 you need to get something in the air that 512 00:30:17,720 --> 00:30:19,580 can react with hydrogen peroxide that they 513 00:30:19,580 --> 00:30:21,410 are fast to remove it. 514 00:30:21,410 --> 00:30:24,690 And then you want to reset your protein, 515 00:30:24,690 --> 00:30:26,690 so it can react with another molecule, 516 00:30:26,690 --> 00:30:29,180 so you need a reductant. 517 00:30:29,180 --> 00:30:32,560 So these are the key-- 518 00:30:32,560 --> 00:30:36,730 for two of the four things I was going 519 00:30:36,730 --> 00:30:39,710 to describe in terms of defense. 520 00:30:39,710 --> 00:30:44,540 Another one is catalase, this is the one that if you go back 521 00:30:44,540 --> 00:30:47,300 and you look at the Carroll paper, which we discussed 522 00:30:47,300 --> 00:30:55,400 actually in class, these are heme proteins, 523 00:30:55,400 --> 00:30:58,790 and these are distinct from the myeloperoxidase 524 00:30:58,790 --> 00:31:03,170 that we'll talk about in this section. 525 00:31:03,170 --> 00:31:06,890 But they can take hydrogen peroxide, 526 00:31:06,890 --> 00:31:11,700 and they can convert it to oxygen plus water. 527 00:31:11,700 --> 00:31:17,300 So what they've done is removed a putative reactant species. 528 00:31:17,300 --> 00:31:20,630 Again, how reactive it is depends on the environment, 529 00:31:20,630 --> 00:31:22,430 and turn it back into oxygen and water, 530 00:31:22,430 --> 00:31:25,760 which are completely unreactive. 531 00:31:25,760 --> 00:31:30,050 And the fourth, which is used quite frequently, 532 00:31:30,050 --> 00:31:33,530 are the glutathione peroxidases. 533 00:31:33,530 --> 00:31:34,520 And this is the one-- 534 00:31:37,940 --> 00:31:40,820 I just told you what the structure of glutathione 535 00:31:40,820 --> 00:31:42,320 is, peroxidase is. 536 00:31:45,430 --> 00:31:47,590 You all know from the Carroll paper 537 00:31:47,590 --> 00:31:49,750 that you have a single reactive cystine 538 00:31:49,750 --> 00:31:51,820 in glutathione peroxidase 3. 539 00:31:51,820 --> 00:31:56,800 That's what we used as the model for all of our redox chemistry. 540 00:31:56,800 --> 00:31:58,990 Some of the glutathione peroxidases 541 00:31:58,990 --> 00:32:00,820 actually use selenium. 542 00:32:00,820 --> 00:32:03,190 So there's the 20 second amino acid 543 00:32:03,190 --> 00:32:08,200 is selenocysteine This is one of the few enzymes, 544 00:32:08,200 --> 00:32:10,570 as our thioredoxin reductase, which 545 00:32:10,570 --> 00:32:15,130 are involved in this whole redox balance system, 546 00:32:15,130 --> 00:32:17,260 are selenoproteins. 547 00:32:17,260 --> 00:32:19,160 We're not going to talk about those. 548 00:32:19,160 --> 00:32:24,330 But the glutathione peroxidases is actually take 549 00:32:24,330 --> 00:32:28,470 two molecules of glutathione plus hydrogen peroxide. 550 00:32:28,470 --> 00:32:32,370 Again, there are many, many isozymes, 551 00:32:32,370 --> 00:32:37,410 and they can oxidize this to the oxidized form. 552 00:32:37,410 --> 00:32:39,802 So this is the reduced form. 553 00:32:39,802 --> 00:32:41,820 You have a cystine. 554 00:32:41,820 --> 00:32:44,820 And this is the oxidized form. 555 00:32:44,820 --> 00:32:46,860 So you have a disulfide. 556 00:32:46,860 --> 00:32:52,050 Now, again, where have you seen this thiol disulfide system 557 00:32:52,050 --> 00:32:54,160 before? 558 00:32:54,160 --> 00:32:55,780 I mean, bacteria have these things. 559 00:32:55,780 --> 00:32:59,380 We're talking now we're focused on human systems. 560 00:32:59,380 --> 00:33:03,640 Do you remember what happens in the periplasm bacteria? 561 00:33:03,640 --> 00:33:06,280 Did you talk about that this year? 562 00:33:06,280 --> 00:33:08,290 So you haven't seen this before in past. 563 00:33:08,290 --> 00:33:12,730 So bacteria in the periplasm enzymes 564 00:33:12,730 --> 00:33:15,160 that control what thiols you have 565 00:33:15,160 --> 00:33:17,720 and the state of the thiol. 566 00:33:17,720 --> 00:33:22,180 So this redox balance by this disulfide interchange, 567 00:33:22,180 --> 00:33:27,520 very similar to this kind of chemistry, is everywhere. 568 00:33:27,520 --> 00:33:29,560 And the chemistry is pretty simple. 569 00:33:29,560 --> 00:33:36,790 But if you go from cystine to a disulfide, 570 00:33:36,790 --> 00:33:38,330 you just don't go there. 571 00:33:38,330 --> 00:33:40,060 You just don't go there with oxygen. 572 00:33:40,060 --> 00:33:41,890 I think this is something that people get 573 00:33:41,890 --> 00:33:44,350 confused about all the time. 574 00:33:44,350 --> 00:33:48,220 You're doing an oxidation, something has to be reduced. 575 00:33:48,220 --> 00:33:51,300 So hydrogen peroxide, [INAUDIBLE],, 576 00:33:51,300 --> 00:33:53,170 you go through sulfenic acid. 577 00:33:53,170 --> 00:33:57,280 You can now picture that you can have general acid catalyze, 578 00:33:57,280 --> 00:34:01,570 general base catalyze, cleavage of disulfide bond formation. 579 00:34:01,570 --> 00:34:03,940 So just because you have oxygen around 580 00:34:03,940 --> 00:34:07,540 and reduced cystines around doesn't mean you automatically 581 00:34:07,540 --> 00:34:10,810 rapidly have disulfides around. 582 00:34:10,810 --> 00:34:13,429 Again, you need to think about the chemistry of what's 583 00:34:13,429 --> 00:34:13,929 going on. 584 00:34:16,850 --> 00:34:19,090 So now what I want to do is then show you 585 00:34:19,090 --> 00:34:22,570 sort of the general model. 586 00:34:22,570 --> 00:34:26,659 And then we'll talk about the NADPH oxidases, 587 00:34:26,659 --> 00:34:31,270 which is the focus of module 7. 588 00:34:31,270 --> 00:34:33,260 The general model is as follows. 589 00:34:33,260 --> 00:34:37,000 So you have an oxygen. And we have the N-- 590 00:34:40,130 --> 00:34:40,750 sorry. 591 00:34:40,750 --> 00:34:42,670 The proteins we're going to be talking about 592 00:34:42,670 --> 00:34:47,302 are NOx2 or another NOx isozyme. 593 00:34:47,302 --> 00:34:48,969 OK, I'm not going to write out the name. 594 00:34:48,969 --> 00:34:54,850 We talked about that in the last recitation section. 595 00:34:54,850 --> 00:34:58,720 These enzymes use-- and I think this is important 596 00:34:58,720 --> 00:35:00,520 because part of the redox switches 597 00:35:00,520 --> 00:35:02,470 that I think are under appreciated 598 00:35:02,470 --> 00:35:04,990 are the levels of NADPH, NADP. 599 00:35:04,990 --> 00:35:09,070 They play incredibly important roles inside the cell. 600 00:35:09,070 --> 00:35:13,645 So you have NADPH going to NADP. 601 00:35:18,190 --> 00:35:19,840 And we talked about the fact-- 602 00:35:19,840 --> 00:35:22,810 and we'll come back to this that this protein 603 00:35:22,810 --> 00:35:25,750 has a flavin and two hemes. 604 00:35:25,750 --> 00:35:30,040 And it produces superoxide 605 00:35:30,040 --> 00:35:32,830 So this is incredibly unusual. 606 00:35:32,830 --> 00:35:35,530 Superoxide is usually an artifact 607 00:35:35,530 --> 00:35:37,660 of some uncoupling reaction that happens 608 00:35:37,660 --> 00:35:39,790 all the time inside the cell. 609 00:35:39,790 --> 00:35:44,680 This enzyme is a professional superoxide generator. 610 00:35:44,680 --> 00:35:45,940 That's what its goal is. 611 00:35:45,940 --> 00:35:48,940 OK, most other times you see superoxide something 612 00:35:48,940 --> 00:35:50,810 has gone astray. 613 00:35:50,810 --> 00:35:57,130 So this is a professional superoxide generator. 614 00:36:00,930 --> 00:36:05,580 And so what happens then, when you generate superoxide 615 00:36:05,580 --> 00:36:10,840 you could have SOD, or you could have protons. 616 00:36:10,840 --> 00:36:15,390 So if you're in a place where the pH is slightly lower, 617 00:36:15,390 --> 00:36:20,420 you generate rapidly, very rapidly hydrogen peroxide. 618 00:36:20,420 --> 00:36:25,020 OK, so superoxide doesn't sit around all that long. 619 00:36:25,020 --> 00:36:34,200 If you have iron 3 around, it could be bound to something. 620 00:36:34,200 --> 00:36:40,780 What happens is the superoxide combines with the iron 3 621 00:36:40,780 --> 00:36:49,220 to reduce it to iron 2 plus oxygen. 622 00:36:49,220 --> 00:36:51,740 So superoxide if you look at the reduction potentials, 623 00:36:51,740 --> 00:36:53,780 obviously what does it depend on? 624 00:36:53,780 --> 00:36:56,850 It depends on the ligand environment of the iron 3. 625 00:36:56,850 --> 00:36:58,400 That affects the redox potential. 626 00:36:58,400 --> 00:37:01,490 Hopefully, you all know that and have 627 00:37:01,490 --> 00:37:04,980 thought about that at this stage, given the last module. 628 00:37:04,980 --> 00:37:10,790 So what happens now is the hydrogen peroxide 629 00:37:10,790 --> 00:37:13,160 can react with iron 2. 630 00:37:13,160 --> 00:37:14,570 And this is the killer. 631 00:37:14,570 --> 00:37:17,180 That does what's called Fenton's chemistry, which 632 00:37:17,180 --> 00:37:19,830 generates hydroxide radical. 633 00:37:19,830 --> 00:37:26,180 So these two guys, hydrogen peroxide and iron, 634 00:37:26,180 --> 00:37:33,890 now combine by what is called, in the review, Fenton's 635 00:37:33,890 --> 00:37:37,040 chemistry. 636 00:37:37,040 --> 00:37:41,570 And I'm not going to write out the detailed mechanism of how 637 00:37:41,570 --> 00:37:43,760 this works in fact, I think we still really 638 00:37:43,760 --> 00:37:45,710 don't completely understand it. 639 00:37:45,710 --> 00:37:49,130 But anyhow, you're generating this reactive species, 640 00:37:49,130 --> 00:37:53,630 hydroxide radical, which is dying to be reduced. 641 00:37:53,630 --> 00:37:56,485 So this guy is responsible. 642 00:37:56,485 --> 00:37:59,310 It hits anything, and it reacts. 643 00:37:59,310 --> 00:38:04,400 So it ultimately is responsible for modifying lipids, modifying 644 00:38:04,400 --> 00:38:08,360 sugars, modifying amino acids, modifying nucleic acid. 645 00:38:08,360 --> 00:38:12,410 This guy-- because it's so reactive-- 646 00:38:12,410 --> 00:38:23,450 damages proteins, DNA, RNA, I'm not going 647 00:38:23,450 --> 00:38:25,160 to write all this out, lipids. 648 00:38:25,160 --> 00:38:26,770 This is the guy. 649 00:38:26,770 --> 00:38:31,780 And that's described in the review article you had to read. 650 00:38:31,780 --> 00:38:35,110 And what can this hydrogen peroxide also do? 651 00:38:35,110 --> 00:38:37,580 We'll also see that the hydrogen peroxide, 652 00:38:37,580 --> 00:38:43,980 which is going to be generated inside the neutrophil, which 653 00:38:43,980 --> 00:38:47,310 we're going to be focusing on, the white blood cells that 654 00:38:47,310 --> 00:38:50,040 are going to be trying to take care of the bacteria 655 00:38:50,040 --> 00:38:55,500 or viruses, and that you have chloride. 656 00:38:55,500 --> 00:38:57,480 Now, you form hypochlorous acid. 657 00:39:00,560 --> 00:39:04,030 So these are the kinds of guys, HO dot, 658 00:39:04,030 --> 00:39:09,190 HOCl are guys that are going to actually do destructive things 659 00:39:09,190 --> 00:39:14,200 when they react with things that help us to defend ourselves 660 00:39:14,200 --> 00:39:17,260 against bacterial insults. 661 00:39:17,260 --> 00:39:21,270 So that's a picture of the big overview. 662 00:39:21,270 --> 00:39:25,080 And so that's that and we're going to be simply focusing 663 00:39:25,080 --> 00:39:26,498 on two proteins. 664 00:39:26,498 --> 00:39:28,290 The first protein we're going to talk about 665 00:39:28,290 --> 00:39:32,550 is the one we went through in recitation, NOx2. 666 00:39:32,550 --> 00:39:35,250 And I'm not going to write down that reaction. 667 00:39:35,250 --> 00:39:38,340 Hopefully you all know this by now. 668 00:39:38,340 --> 00:39:40,800 I just sort of said that over there. 669 00:39:40,800 --> 00:39:44,040 And there are 11 different isozymes, and then 670 00:39:44,040 --> 00:39:46,500 myeloperoxidase, which both of these 671 00:39:46,500 --> 00:39:51,690 are found in the neutrophils in the phagosome 672 00:39:51,690 --> 00:39:52,600 of the neutrophils. 673 00:40:00,670 --> 00:40:03,100 OK, so let me-- 674 00:40:03,100 --> 00:40:09,370 so the chemistry that goes on with the NOx proteins 675 00:40:09,370 --> 00:40:10,460 is complicated. 676 00:40:10,460 --> 00:40:12,970 So it's not just the NOx protein. 677 00:40:12,970 --> 00:40:15,010 We're going to talk about the NOx protein. 678 00:40:15,010 --> 00:40:18,122 But as with everything, there are other factors 679 00:40:18,122 --> 00:40:19,580 that play a key role, and I'm going 680 00:40:19,580 --> 00:40:24,280 to show you a cartoon with what the other factors are. 681 00:40:24,280 --> 00:40:26,680 But we're not going to talk about the details of how 682 00:40:26,680 --> 00:40:32,560 those factors help the NOx2 protein make superoxide OK. 683 00:40:32,560 --> 00:40:36,190 Make superoxide in a controlled fashion, 684 00:40:36,190 --> 00:40:38,860 that's the key thing, in a controlled fashion. 685 00:40:38,860 --> 00:40:40,900 So we have a NOx protein. 686 00:40:40,900 --> 00:40:42,520 The only one I do want to look at 687 00:40:42,520 --> 00:40:45,880 is the NOx protein itself, because we're 688 00:40:45,880 --> 00:40:49,480 going to use it not only in this lecture, 689 00:40:49,480 --> 00:40:53,170 but also the lecture of NOx2 proteins in signaling. 690 00:40:53,170 --> 00:40:57,220 So the chemistry is the same in destroying the bacteria 691 00:40:57,220 --> 00:40:58,330 and in signaling. 692 00:40:58,330 --> 00:41:01,810 So you need to know what the protein does. 693 00:41:01,810 --> 00:41:09,820 So if you look at NOx2 NADPH oxidases, what do you know? 694 00:41:09,820 --> 00:41:15,400 They exist in a membrane, and we'll 695 00:41:15,400 --> 00:41:19,810 see this membrane can be the phagosome, 696 00:41:19,810 --> 00:41:23,390 or it can be the plasma membrane, or can be-- 697 00:41:23,390 --> 00:41:27,850 I'm going to show you a cartoon of this-- a vesicle membrane. 698 00:41:27,850 --> 00:41:31,780 These proteins are located in many places inside the cell. 699 00:41:31,780 --> 00:41:37,870 But they all sort of have the same predispositions. 700 00:41:37,870 --> 00:41:44,260 So they have one subunit with a domain that has the FAD on it. 701 00:41:44,260 --> 00:41:50,860 So if we're looking at with the epidermal growth factor, 702 00:41:50,860 --> 00:41:52,990 or if we're looking at the neutrophils, 703 00:41:52,990 --> 00:41:55,630 this would be the cytosol, if we're 704 00:41:55,630 --> 00:41:57,970 looking at the neutrophils, and this 705 00:41:57,970 --> 00:42:03,310 would be the inside the lumen of the phagosome. 706 00:42:07,170 --> 00:42:10,980 And the FAD can be-- 707 00:42:10,980 --> 00:42:12,540 what is the function of FAD? 708 00:42:12,540 --> 00:42:13,960 We've talked about this. 709 00:42:13,960 --> 00:42:19,230 It's a major mediator between two electron chemistry and one 710 00:42:19,230 --> 00:42:21,340 electron chemistry. 711 00:42:21,340 --> 00:42:22,590 And you've seen that before. 712 00:42:22,590 --> 00:42:26,820 Hopefully that was grilled into you in the respiratory chain. 713 00:42:26,820 --> 00:42:30,150 So in the respiratory chain, you have iron sulfur clusters, 714 00:42:30,150 --> 00:42:31,530 or you have hemes. 715 00:42:31,530 --> 00:42:33,300 Here we're going to have hemes. 716 00:42:33,300 --> 00:42:37,770 And so what we have is on this face, 717 00:42:37,770 --> 00:42:44,055 we have NADPH going to NADP plus a proton. 718 00:42:46,590 --> 00:42:49,380 This turns out to be important, because that also 719 00:42:49,380 --> 00:42:53,760 controls the pH, and there are voltage channels controlled 720 00:42:53,760 --> 00:42:55,650 by pH that you need to think about if you 721 00:42:55,650 --> 00:42:58,530 looked at the detail biology. 722 00:42:58,530 --> 00:43:00,810 And so this protein is gp91. 723 00:43:03,420 --> 00:43:09,930 That is it's 91 kilodaltons, and gp means it's a glycoprotein. 724 00:43:09,930 --> 00:43:11,700 And then you have a second protein 725 00:43:11,700 --> 00:43:16,380 that's also an integral membrane protein, that's also critical. 726 00:43:16,380 --> 00:43:17,250 And this is gp22. 727 00:43:20,270 --> 00:43:27,220 And so what you see is you have iron-- 728 00:43:27,220 --> 00:43:33,490 you have two hemes, cytochrome b heme, dependent systems. 729 00:43:33,490 --> 00:43:38,710 And these are going to change redox state. 730 00:43:38,710 --> 00:43:43,750 And the interesting thing is that these two teams 731 00:43:43,750 --> 00:43:46,370 are completely coordinated. 732 00:43:46,370 --> 00:43:49,850 So where have you seen heme before that reversibly binds 733 00:43:49,850 --> 00:43:51,050 oxygen? 734 00:43:51,050 --> 00:43:54,020 We need to do something with oxygen. Oxygen is 735 00:43:54,020 --> 00:43:55,910 getting reduced. 736 00:43:55,910 --> 00:44:01,400 But what I'm telling you is that oxygen does not get 737 00:44:01,400 --> 00:44:04,670 reduced by binding to the heme. 738 00:44:04,670 --> 00:44:07,970 It's going to be using this method of electron transfer 739 00:44:07,970 --> 00:44:08,870 that we talked about. 740 00:44:08,870 --> 00:44:10,245 So they've got to be close enough 741 00:44:10,245 --> 00:44:12,830 so you can do electron transfer, perhaps 742 00:44:12,830 --> 00:44:15,230 through the heme edge in the protein. 743 00:44:15,230 --> 00:44:27,170 So ultimately, oxygen is getting converted into superoxide not 744 00:44:27,170 --> 00:44:35,130 by direct binding to the heme. 745 00:44:35,130 --> 00:44:36,870 So this is distinct. 746 00:44:36,870 --> 00:44:38,310 And we'll see, this is completely 747 00:44:38,310 --> 00:44:40,500 distinct from the myeloperoxidases. 748 00:44:40,500 --> 00:44:43,110 This is completely distinct from the P450s 749 00:44:43,110 --> 00:44:48,090 we alluded to when we're talking about cholesterol homeostasis. 750 00:44:48,090 --> 00:44:50,100 And the key that makes all of this work 751 00:44:50,100 --> 00:44:53,370 is that it can form complexes with other proteins. 752 00:44:53,370 --> 00:44:56,070 So let me just tell you what those other proteins are, 753 00:44:56,070 --> 00:45:01,470 and that was described in some detail in the reading. 754 00:45:01,470 --> 00:45:04,800 So we're going to have a GTPase. 755 00:45:04,800 --> 00:45:07,080 RAC2 is a G-protein. 756 00:45:07,080 --> 00:45:10,950 OK, so G-proteins can mediate phosphorylations. 757 00:45:10,950 --> 00:45:14,310 This one mediates phosphorylations. 758 00:45:14,310 --> 00:45:18,280 And it remains in the inhibited state 759 00:45:18,280 --> 00:45:22,080 till you need to trigger off your signaling cascade 760 00:45:22,080 --> 00:45:23,460 by another protein. 761 00:45:23,460 --> 00:45:26,010 The nomenclature is horrible, but there is an inhibitor 762 00:45:26,010 --> 00:45:30,870 protein that binds to the G-protein making it inactive, 763 00:45:30,870 --> 00:45:33,930 when some sensor comes in, they dissociate, and then 764 00:45:33,930 --> 00:45:35,537 the G-protein can function. 765 00:45:35,537 --> 00:45:37,620 And we're going to look at that kind of signaling. 766 00:45:37,620 --> 00:45:39,620 We already have looked at that kind of signaling 767 00:45:39,620 --> 00:45:41,760 in the Carroll paper. 768 00:45:41,760 --> 00:45:46,500 But we'll look at it again in the signaling by NOx. 769 00:45:46,500 --> 00:45:48,840 The second group of proteins-- again, 770 00:45:48,840 --> 00:45:50,160 they're based on this size. 771 00:45:50,160 --> 00:45:52,430 These were identified a long time ago. 772 00:45:52,430 --> 00:45:54,570 They are unique to the phagosome. 773 00:45:54,570 --> 00:45:56,340 They're called phagosome oxidases. 774 00:45:56,340 --> 00:45:58,380 That's going to be the organelle where we're 775 00:45:58,380 --> 00:46:01,510 going to kill the bacteria. 776 00:46:01,510 --> 00:46:05,510 And so this p47 needs to be phosphorylated 777 00:46:05,510 --> 00:46:08,040 and it's phosphorylated by the G-protein. 778 00:46:08,040 --> 00:46:10,320 And that's key to have everything come together 779 00:46:10,320 --> 00:46:11,970 to allow the chemistry happen. 780 00:46:11,970 --> 00:46:14,850 So this chemistry, in this form, it's inactive. 781 00:46:14,850 --> 00:46:17,310 It's only when everything comes together 782 00:46:17,310 --> 00:46:20,170 that you actually start doing the chemistry that's 783 00:46:20,170 --> 00:46:22,650 going to help us. 784 00:46:22,650 --> 00:46:24,930 So here's the model. 785 00:46:24,930 --> 00:46:27,420 So here's a resting cell. 786 00:46:27,420 --> 00:46:29,250 This is the nucleus. 787 00:46:29,250 --> 00:46:33,630 Here, the blue thing is the NOx protein. 788 00:46:33,630 --> 00:46:38,760 And the little blue thing is the second subunit. 789 00:46:38,760 --> 00:46:40,140 This is the 91. 790 00:46:40,140 --> 00:46:41,690 This is the 22. 791 00:46:41,690 --> 00:46:44,670 Here we can see that it's located in the plasma membrane. 792 00:46:44,670 --> 00:46:45,870 That's one of its locations. 793 00:46:45,870 --> 00:46:49,410 That's not the predominant location in the resting state. 794 00:46:49,410 --> 00:46:51,390 The predominant location apparently 795 00:46:51,390 --> 00:46:54,600 is in little vesicles within these neutrophils, 796 00:46:54,600 --> 00:46:57,900 the white blood cells that are the first defenders 797 00:46:57,900 --> 00:47:01,990 against invasion by bacterial systems. 798 00:47:01,990 --> 00:47:04,730 And then we have these little complexes. 799 00:47:04,730 --> 00:47:08,550 Here's RAC2, a GTPase that's inhibited. 800 00:47:08,550 --> 00:47:12,560 And here is the phagosome oxidase. 801 00:47:12,560 --> 00:47:16,350 And then what happens when the bacteria comes in, 802 00:47:16,350 --> 00:47:22,020 somehow the bacteria is coming in over here, it gets engulfed, 803 00:47:22,020 --> 00:47:26,490 and you form these little phagosomes inside the cell. 804 00:47:26,490 --> 00:47:30,790 And now what happens is the NADPH, 805 00:47:30,790 --> 00:47:33,900 the NOx proteins are located like this. 806 00:47:33,900 --> 00:47:36,420 The NADPH is on the outside. 807 00:47:36,420 --> 00:47:41,550 It's been activated by this GTPase. 808 00:47:41,550 --> 00:47:47,130 And now it's ready to generate superoxide inside the cell. 809 00:47:47,130 --> 00:47:50,010 So there's a lot of membrane fusion and reorganization. 810 00:47:50,010 --> 00:47:52,530 Obviously, the signaling is really complex. 811 00:47:52,530 --> 00:47:54,310 We know a lot about the signaling. 812 00:47:54,310 --> 00:47:58,260 How do these guys even know there's a bacteria out there? 813 00:47:58,260 --> 00:48:00,710 How do they sense all of that? 814 00:48:00,710 --> 00:48:02,460 And I don't know if this is going to work. 815 00:48:02,460 --> 00:48:07,050 But this is a sort of a cool picture if it does work. 816 00:48:07,050 --> 00:48:09,982 Although it worked in my office, but it might not work here. 817 00:48:09,982 --> 00:48:10,690 Oh, here it goes. 818 00:48:10,690 --> 00:48:12,060 So here we are. 819 00:48:12,060 --> 00:48:14,190 This is a white blood cell. 820 00:48:14,190 --> 00:48:15,960 These are red blood cells. 821 00:48:15,960 --> 00:48:18,660 The bacteria, these little things, floating around. 822 00:48:18,660 --> 00:48:20,580 It's sending off a signal. 823 00:48:20,580 --> 00:48:24,900 The white blood cell is chasing the bacteria. 824 00:48:24,900 --> 00:48:26,730 So there's some sequences chasing it 825 00:48:26,730 --> 00:48:28,500 through all of these cells, and you're 826 00:48:28,500 --> 00:48:30,330 going to see that in a minute, it gets it. 827 00:48:30,330 --> 00:48:30,930 There it goes. 828 00:48:30,930 --> 00:48:32,100 Gets inside. 829 00:48:32,100 --> 00:48:35,910 It's now in the phagosome, and puff, everything disappears. 830 00:48:35,910 --> 00:48:38,660 And that is really what's going on in the system. 831 00:48:38,660 --> 00:48:39,810 So the question is-- 832 00:48:39,810 --> 00:48:41,280 it's a really cool picture. 833 00:48:41,280 --> 00:48:43,620 The question is what's the chemistry that's actually 834 00:48:43,620 --> 00:48:45,075 going on in these systems. 835 00:48:47,880 --> 00:48:51,670 So the chemistry-- whoops, somehow I lost-- 836 00:48:51,670 --> 00:48:53,010 I'm already over. 837 00:48:53,010 --> 00:48:55,460 That chemistry, it's all over already. 838 00:48:55,460 --> 00:48:58,490 What we'll do next time is come back 839 00:48:58,490 --> 00:49:01,040 and talk about how this flavin works, 840 00:49:01,040 --> 00:49:03,790 and then we'll see in that little phagosome also 841 00:49:03,790 --> 00:49:05,450 is a myeloperoxidase. 842 00:49:05,450 --> 00:49:07,413 We'll talk about how that works, and those 843 00:49:07,413 --> 00:49:09,080 are the two things I want-- how did they 844 00:49:09,080 --> 00:49:11,090 degrade this bacteria once they get 845 00:49:11,090 --> 00:49:15,000 inside this little organelle?