1 00:00:00,500 --> 00:00:02,820 The following content is provided under a Creative 2 00:00:02,820 --> 00:00:04,360 Commons license. 3 00:00:04,360 --> 00:00:06,660 Your support will help MIT OpenCourseWare 4 00:00:06,660 --> 00:00:11,020 continue to offer high quality educational resources for free. 5 00:00:11,020 --> 00:00:13,650 To make a donation or view additional materials 6 00:00:13,650 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,550 at ocw.mit.edu. 8 00:00:25,740 --> 00:00:27,240 JOANNE STUBBE: So that's where we're 9 00:00:27,240 --> 00:00:29,760 going in terms of Module Six. 10 00:00:29,760 --> 00:00:32,340 We introduced this the last time. 11 00:00:32,340 --> 00:00:36,750 And this is the required reading that's also been posted-- 12 00:00:36,750 --> 00:00:38,580 and we started this last time. 13 00:00:41,380 --> 00:00:44,130 This introductory lecture is talking about metals, 14 00:00:44,130 --> 00:00:47,730 in general-- the chemical properties of metals, 15 00:00:47,730 --> 00:00:51,660 and why we have these kinds of metals in our bodies. 16 00:00:51,660 --> 00:00:54,480 Why we're using these kinds of metals. 17 00:00:54,480 --> 00:01:00,600 And focusing then on metal homeostasis, in general. 18 00:01:00,600 --> 00:01:03,690 And then we will move over into complete focus 19 00:01:03,690 --> 00:01:07,030 on iron for the next three-- 20 00:01:07,030 --> 00:01:08,630 so, subsequent three lectures. 21 00:01:08,630 --> 00:01:09,240 OK. 22 00:01:09,240 --> 00:01:11,640 So here we are-- 23 00:01:11,640 --> 00:01:13,440 here we are with the periodic table. 24 00:01:13,440 --> 00:01:17,490 And we're going to be focusing on transition-- 25 00:01:17,490 --> 00:01:20,670 the transition metals, which are most 26 00:01:20,670 --> 00:01:23,730 of the places where you see the chemistry 27 00:01:23,730 --> 00:01:26,040 that you're familiar with. 28 00:01:26,040 --> 00:01:28,950 And this just sort of tells you the relative abundance 29 00:01:28,950 --> 00:01:31,820 of the metals in our bodies. 30 00:01:31,820 --> 00:01:32,580 OK? 31 00:01:32,580 --> 00:01:35,970 And we talked about last time-- 32 00:01:35,970 --> 00:01:40,650 sort of an introduction to the different kinds of chemistries 33 00:01:40,650 --> 00:01:41,580 that we can have. 34 00:01:41,580 --> 00:01:46,530 And we talked about iron transport, reversible oxygen 35 00:01:46,530 --> 00:01:52,530 binding, and then we were at the place for electron transfer 36 00:01:52,530 --> 00:01:55,410 with nitrogen fixation. 37 00:01:55,410 --> 00:01:59,280 Again, this is a cursory overview with signaling, 38 00:01:59,280 --> 00:02:01,538 where I introduced the fact that you have calcium. 39 00:02:01,538 --> 00:02:04,080 And also you can have zinc and copper signaling, which people 40 00:02:04,080 --> 00:02:06,510 didn't realize until recently. 41 00:02:06,510 --> 00:02:11,160 Zinc is worked on extensively by the Lippard Lab and copper 42 00:02:11,160 --> 00:02:15,380 is worked on by other people-- the Chang Lab at Berkeley 43 00:02:15,380 --> 00:02:17,130 We will see we're going to have regulation 44 00:02:17,130 --> 00:02:19,297 at the transcriptional and the translational level-- 45 00:02:19,297 --> 00:02:20,520 that's true for all metals-- 46 00:02:20,520 --> 00:02:23,010 and that many kinds of reactions can happen. 47 00:02:23,010 --> 00:02:26,460 I'm only going to focus on the reactions that we're going-- 48 00:02:26,460 --> 00:02:33,390 that are related to iron in the course of this module. 49 00:02:33,390 --> 00:02:35,700 We had gone through reversible oxygen binding, 50 00:02:35,700 --> 00:02:37,200 and at the end of the last lecture 51 00:02:37,200 --> 00:02:43,900 we were focused on the amazing diversity of metallocofactors. 52 00:02:43,900 --> 00:02:46,710 These are some of my favorite metallocofactors. 53 00:02:46,710 --> 00:02:49,290 Most of you, I think, are not exposed to this. 54 00:02:49,290 --> 00:02:52,080 You sort of know there's an interesting cofactor, 55 00:02:52,080 --> 00:02:55,870 but I haven't really thought about how these cofactors work. 56 00:02:55,870 --> 00:02:58,380 And if you look at this one-- at the end, 57 00:02:58,380 --> 00:03:01,140 we were talking about-- you have-- 58 00:03:01,140 --> 00:03:04,050 this is the active cofactor formed-- 59 00:03:04,050 --> 00:03:06,390 found in the enzyme nitrogenase, which 60 00:03:06,390 --> 00:03:11,250 does an eight-electron reduction of nitrogen 61 00:03:11,250 --> 00:03:13,760 to ammonia plus hydrogen. 62 00:03:13,760 --> 00:03:16,770 And there are many enzymatic systems 63 00:03:16,770 --> 00:03:19,290 that use multi electrons, OK? 64 00:03:19,290 --> 00:03:22,680 And that's an active area from the chemical point of view, 65 00:03:22,680 --> 00:03:23,190 as well. 66 00:03:23,190 --> 00:03:26,550 How do you control multi electron oxidation 67 00:03:26,550 --> 00:03:28,290 and reduction, and what is the multi 68 00:03:28,290 --> 00:03:31,770 electrons versus single electrons get you? 69 00:03:31,770 --> 00:03:36,150 That's a hot area of chemistry now in the bio-inorganic world. 70 00:03:36,150 --> 00:03:39,540 And I think most intriguing is this little carbon 71 00:03:39,540 --> 00:03:40,290 in the middle. 72 00:03:40,290 --> 00:03:41,353 That's a carbon minus 4. 73 00:03:41,353 --> 00:03:42,270 How does it get there? 74 00:03:42,270 --> 00:03:43,270 Where does it come from? 75 00:03:43,270 --> 00:03:44,880 That should intrigue you and-- 76 00:03:44,880 --> 00:03:47,430 actually, where is even the reactive species? 77 00:03:47,430 --> 00:03:52,350 Where does nitrogen bind to do the reduction? 78 00:03:52,350 --> 00:03:55,590 Hydrogenase is another active area of research, 79 00:03:55,590 --> 00:03:57,000 now-- in energy. 80 00:03:57,000 --> 00:03:59,250 People thinking about how do you do-- 81 00:03:59,250 --> 00:04:02,730 how do you use catalysts to do oxygen evolution, here? 82 00:04:02,730 --> 00:04:06,270 Or hydrogen reduction or oxidation? 83 00:04:06,270 --> 00:04:10,140 And people who have taken inspiration from enzymes 84 00:04:10,140 --> 00:04:12,780 called hydrogenases, and they come in many flavors. 85 00:04:12,780 --> 00:04:17,220 You have nickel iron, iron iron, iron only. 86 00:04:17,220 --> 00:04:22,410 And we know quite a bit about the actual chemistry. 87 00:04:22,410 --> 00:04:25,500 The rate constants for turnover are amazingly fast, 88 00:04:25,500 --> 00:04:28,710 and so people are trying to do that in little devices 89 00:04:28,710 --> 00:04:32,250 nowadays, using this as an inspiration to generate 90 00:04:32,250 --> 00:04:34,590 these kinds of catalysts. 91 00:04:34,590 --> 00:04:36,990 And what do you see unusual about this iron cluster? 92 00:04:36,990 --> 00:04:38,760 I'm digressing, but I think this is 93 00:04:38,760 --> 00:04:40,710 a good thing for you to know. 94 00:04:40,710 --> 00:04:42,850 I wouldn't expect you to remember the details, 95 00:04:42,850 --> 00:04:45,330 but what's unusual about this cluster? 96 00:04:45,330 --> 00:04:47,510 Anybody see anything from a chemical perspective 97 00:04:47,510 --> 00:04:48,135 that's unusual? 98 00:04:50,785 --> 00:04:52,917 AUDIENCE: You mean, like all of the ligands on-- 99 00:04:52,917 --> 00:04:53,750 JOANNE STUBBE: Yeah. 100 00:04:53,750 --> 00:04:54,800 Look at the ligands. 101 00:04:54,800 --> 00:04:57,480 What's unusual ? 102 00:04:57,480 --> 00:04:59,360 AUDIENCE: Is there a carbon with five bonds? 103 00:04:59,360 --> 00:05:00,527 JOANNE STUBBE: There a what? 104 00:05:00,527 --> 00:05:03,230 AUDIENCE: Carbon with five bonds? 105 00:05:03,230 --> 00:05:06,290 JOANNE STUBBE: I don't see any carbons with five bonds. 106 00:05:06,290 --> 00:05:06,980 OK, yeah. 107 00:05:06,980 --> 00:05:07,550 So, OK. 108 00:05:07,550 --> 00:05:09,223 That's not what I want you-- so again, 109 00:05:09,223 --> 00:05:10,640 it depends on what the bonding is. 110 00:05:10,640 --> 00:05:13,610 But what is unusual about the carbon with five bonds? 111 00:05:17,678 --> 00:05:19,237 AUDIENCE: It's attached to both. 112 00:05:19,237 --> 00:05:20,070 JOANNE STUBBE: Yeah. 113 00:05:20,070 --> 00:05:21,600 So that's not what I mean, though. 114 00:05:21,600 --> 00:05:23,316 What is unusual about the ligand? 115 00:05:23,316 --> 00:05:24,230 AUDIENCE: It's carbon monoxide. 116 00:05:24,230 --> 00:05:25,980 JOANNE STUBBE: Yeah, it's carbon monoxide. 117 00:05:25,980 --> 00:05:28,050 What do you know about carbon monoxide? 118 00:05:28,050 --> 00:05:29,080 It kills you, right? 119 00:05:29,080 --> 00:05:29,580 OK. 120 00:05:29,580 --> 00:05:33,090 So how do how the heck do we have organisms that have 121 00:05:33,090 --> 00:05:35,100 carbon monoxide ligands, right? 122 00:05:35,100 --> 00:05:37,770 And we all have carbon monoxide detectors in our house 123 00:05:37,770 --> 00:05:40,470 because it binds to our heme proteins and kills us. 124 00:05:40,470 --> 00:05:43,080 What's the other thing that's unusual about 125 00:05:43,080 --> 00:05:44,610 the ligand environment, here? 126 00:05:44,610 --> 00:05:46,350 What's the other ligand that's unusual? 127 00:05:46,350 --> 00:05:47,200 AUDIENCE: Cyanide. 128 00:05:47,200 --> 00:05:48,158 JOANNE STUBBE: Cyanide. 129 00:05:48,158 --> 00:05:49,470 That also kills you. 130 00:05:49,470 --> 00:05:51,960 So automatically as chemists, you 131 00:05:51,960 --> 00:05:54,690 ought to be intrigued by where the heck did these things come 132 00:05:54,690 --> 00:05:57,990 from, and how do you prevent it from killing the organism? 133 00:05:57,990 --> 00:06:01,560 At the same time, is this able to use these ligands 134 00:06:01,560 --> 00:06:03,070 to actually do chemistry? 135 00:06:03,070 --> 00:06:05,940 And if you think about transition metal chemistry-- 136 00:06:05,940 --> 00:06:08,490 we won't talk about this, but we will see-- 137 00:06:08,490 --> 00:06:10,470 what are the oxidation states of iron 138 00:06:10,470 --> 00:06:12,230 that you're most familiar with? 139 00:06:12,230 --> 00:06:13,252 AUDIENCE: Two, three-- 140 00:06:13,252 --> 00:06:14,460 JOANNE STUBBE: Two and three. 141 00:06:14,460 --> 00:06:15,000 OK? 142 00:06:15,000 --> 00:06:18,030 And then what we'll see is four happens transiently, 143 00:06:18,030 --> 00:06:20,670 but we also go to iron zero's. 144 00:06:20,670 --> 00:06:24,570 So we have a wide range of redox spanning chemistry 145 00:06:24,570 --> 00:06:25,800 by altering the ligands. 146 00:06:25,800 --> 00:06:27,967 And that's going to be one of the take home messages 147 00:06:27,967 --> 00:06:29,910 from the four lectures. 148 00:06:29,910 --> 00:06:31,050 OK? 149 00:06:31,050 --> 00:06:33,300 And this is, I think, totally amazing. 150 00:06:33,300 --> 00:06:35,850 We now have an atomic resolution structure 151 00:06:35,850 --> 00:06:37,740 without the metals being destroyed, 152 00:06:37,740 --> 00:06:42,450 which normally happens when you put a metal into an X-ray beam. 153 00:06:42,450 --> 00:06:44,275 The electrons reduce the metal and you 154 00:06:44,275 --> 00:06:45,900 don't end up with the cluster you think 155 00:06:45,900 --> 00:06:47,350 you're going to be getting. 156 00:06:47,350 --> 00:06:49,020 And what you hear see here is you have 157 00:06:49,020 --> 00:06:53,040 four manganeses and a calcium. 158 00:06:53,040 --> 00:06:57,960 And again, this is a multi electron process 159 00:06:57,960 --> 00:07:01,140 where, in order to go from water to oxygen uphill, 160 00:07:01,140 --> 00:07:02,490 you need to have light. 161 00:07:02,490 --> 00:07:05,640 And a lot of people are focused on that now, 162 00:07:05,640 --> 00:07:11,100 in terms of energy production and chemical catalysts 163 00:07:11,100 --> 00:07:13,572 that can mimic these kinds of reactions. 164 00:07:13,572 --> 00:07:15,030 But what we're going to be focusing 165 00:07:15,030 --> 00:07:19,050 on now in the case of the iron is the iron cofactors. 166 00:07:19,050 --> 00:07:22,950 And most of you have seen these iron cofactors before. 167 00:07:22,950 --> 00:07:27,150 This is just a few of the iron sulfur cofactors. 168 00:07:27,150 --> 00:07:28,710 Where have you seen them before? 169 00:07:31,650 --> 00:07:34,455 What part of biochemistry in your introductory courses 170 00:07:34,455 --> 00:07:35,580 have you seen these before? 171 00:07:35,580 --> 00:07:37,955 And then I'll tell you where we're going to see it again. 172 00:07:41,790 --> 00:07:43,390 Nobody has ever seen them before? 173 00:07:46,970 --> 00:07:47,470 No? 174 00:07:47,470 --> 00:07:49,335 AUDIENCE: A lot of single-- a lot of single electron 175 00:07:49,335 --> 00:07:49,640 transfer-- 176 00:07:49,640 --> 00:07:50,080 JOANNE STUBBE: Yeah. 177 00:07:50,080 --> 00:07:51,700 So it's single electron transfer. 178 00:07:51,700 --> 00:07:52,450 Where? 179 00:07:52,450 --> 00:07:53,260 In respiration. 180 00:07:53,260 --> 00:07:56,320 Hopefully you all did that as a basic introductory part. 181 00:07:56,320 --> 00:07:59,050 You have iron-sulfur clusters all over the place, 182 00:07:59,050 --> 00:08:00,760 and these are the clusters that were 183 00:08:00,760 --> 00:08:02,590 found in the prebiotic world. 184 00:08:02,590 --> 00:08:05,920 So they are incredibly interesting. 185 00:08:05,920 --> 00:08:07,760 What we're going to be focused on 186 00:08:07,760 --> 00:08:10,270 are four iron, four sulfur clusters. 187 00:08:10,270 --> 00:08:14,290 That's a key-- that's a key component that allows 188 00:08:14,290 --> 00:08:17,710 us to sense iron in humans. 189 00:08:17,710 --> 00:08:19,750 And so we'll come back to the four iron, 190 00:08:19,750 --> 00:08:22,600 four sulfur cluster later on. 191 00:08:22,600 --> 00:08:23,620 And all of these-- 192 00:08:23,620 --> 00:08:25,370 I just leave you to think about, where 193 00:08:25,370 --> 00:08:26,650 do these things come from? 194 00:08:26,650 --> 00:08:29,140 You just think you throw in iron and molybdenum 195 00:08:29,140 --> 00:08:32,990 and you have a cofactor that looks like that? 196 00:08:32,990 --> 00:08:34,365 The answer is no. 197 00:08:34,365 --> 00:08:36,490 The other thing that I just wanted to introduce you 198 00:08:36,490 --> 00:08:40,570 to because this is a very active area of research 199 00:08:40,570 --> 00:08:43,360 in our department-- the Drennan lab-- 200 00:08:43,360 --> 00:08:44,920 and a lot of my former students have 201 00:08:44,920 --> 00:08:49,540 been working on enzymes called radical SAM enzymes. 202 00:08:49,540 --> 00:08:51,730 What does SAM normally do? 203 00:08:51,730 --> 00:08:53,620 S-Adenosyl mithionine? 204 00:08:53,620 --> 00:08:56,380 What does that normally do in biology? 205 00:09:02,947 --> 00:09:04,530 AUDIENCE: Does it methylate something? 206 00:09:04,530 --> 00:09:05,650 JOANNE STUBBE: Yeah, it methylates. 207 00:09:05,650 --> 00:09:07,540 So, you know-- here is that you have something that's 208 00:09:07,540 --> 00:09:09,400 activated for nucleophilic attack-- that's 209 00:09:09,400 --> 00:09:10,600 what it normally does. 210 00:09:10,600 --> 00:09:15,850 We now know there are 130,000 reactions that involve SAM 211 00:09:15,850 --> 00:09:18,010 that doesn't do a methylation. 212 00:09:18,010 --> 00:09:25,060 You do reductive cleavage of the carbon methyl bond 213 00:09:25,060 --> 00:09:27,340 to form this carbon methyl-- 214 00:09:27,340 --> 00:09:29,860 carbon sulfur bond to generate a radical. 215 00:09:29,860 --> 00:09:32,680 And you do really complex free radical chemistry. 216 00:09:32,680 --> 00:09:36,700 For example, 50% of all the methane gas in the environment 217 00:09:36,700 --> 00:09:39,550 comes from a radical SAM enzyme that cleaves the phosphorus 218 00:09:39,550 --> 00:09:41,410 carbon bond. 219 00:09:41,410 --> 00:09:45,990 If you look at the antibiotic resistance problem we have now, 220 00:09:45,990 --> 00:09:48,310 there's methylation in the active site-- 221 00:09:48,310 --> 00:09:52,000 the A site of the ribosome that you guys talk 222 00:09:52,000 --> 00:09:55,000 about-- that prevents five different antibiotics 223 00:09:55,000 --> 00:09:56,150 from binding. 224 00:09:56,150 --> 00:09:57,970 And it doesn't involve methylation 225 00:09:57,970 --> 00:09:59,500 in the standard form, it involves 226 00:09:59,500 --> 00:10:01,000 complex free radical chemistry. 227 00:10:01,000 --> 00:10:03,460 So I'm not going to say any more than that, 228 00:10:03,460 --> 00:10:06,400 but radical chemistry is taking off. 229 00:10:06,400 --> 00:10:09,610 I mean, there's all kinds of unusual chemistry that peak 230 00:10:09,610 --> 00:10:13,300 chemists didn't think was possible before, and we're-- 231 00:10:13,300 --> 00:10:15,070 every time we study another system, 232 00:10:15,070 --> 00:10:16,900 we learn something new and exciting 233 00:10:16,900 --> 00:10:18,750 from a chemical perspective. 234 00:10:18,750 --> 00:10:19,473 OK. 235 00:10:19,473 --> 00:10:20,890 So now what I really want to do is 236 00:10:20,890 --> 00:10:23,080 sort of get more into the nitty gritty. 237 00:10:23,080 --> 00:10:24,040 And so one of those-- 238 00:10:24,040 --> 00:10:25,570 I showed you the periodic table. 239 00:10:25,570 --> 00:10:28,330 We have manganese, we have iron, we have copper. 240 00:10:28,330 --> 00:10:29,950 Why were those chosen? 241 00:10:29,950 --> 00:10:35,110 And in part, those are chosen because it reflects-- 242 00:10:35,110 --> 00:10:39,780 the metals reflect earth's history. 243 00:10:45,050 --> 00:10:48,350 And one of the-- 244 00:10:48,350 --> 00:10:52,160 so, one of the things in the geochemical-- 245 00:10:52,160 --> 00:10:54,620 what we know about the geochemical production 246 00:10:54,620 --> 00:11:00,300 of the earth over the eons since its first-- 247 00:11:00,300 --> 00:11:02,840 since a big bang, anyhow. 248 00:11:02,840 --> 00:11:04,550 Here's the earth's core. 249 00:11:04,550 --> 00:11:10,160 And this is taken from the article by Fry and Reed. 250 00:11:10,160 --> 00:11:12,740 And I think it sort of-- this and then the next slide 251 00:11:12,740 --> 00:11:14,000 I'm going to be showing you-- 252 00:11:14,000 --> 00:11:16,670 sort of, I think, places in perspective 253 00:11:16,670 --> 00:11:21,110 why we're using iron and copper and zinc in almost all 254 00:11:21,110 --> 00:11:24,440 the enzymes we see inside of ourselves. 255 00:11:24,440 --> 00:11:28,955 So the earth's core is here. 256 00:11:28,955 --> 00:11:30,830 And then we have-- so we have the inner core, 257 00:11:30,830 --> 00:11:32,240 and we have the outer core. 258 00:11:32,240 --> 00:11:34,690 These two cores are 80% iron. 259 00:11:34,690 --> 00:11:36,080 OK? 260 00:11:36,080 --> 00:11:37,340 We then have the mantle. 261 00:11:37,340 --> 00:11:39,890 And then we have the crust. 262 00:11:39,890 --> 00:11:42,560 And the crust has-- 263 00:11:42,560 --> 00:11:46,370 the fourth most abundant metal is iron. 264 00:11:46,370 --> 00:11:49,880 But you also have other things in the crust-- aluminum, 265 00:11:49,880 --> 00:11:52,970 calcium, silicon. 266 00:11:52,970 --> 00:11:57,650 Why are we using carbon and not silicon, if this is the most-- 267 00:11:57,650 --> 00:12:01,130 most abundant-- one of the most abundant elements 268 00:12:01,130 --> 00:12:02,720 in the earth's core? 269 00:12:02,720 --> 00:12:05,660 And this article sort of goes in and discusses 270 00:12:05,660 --> 00:12:06,628 those kinds of issues. 271 00:12:06,628 --> 00:12:09,170 Making you think about what you learned in freshman chemistry 272 00:12:09,170 --> 00:12:12,500 about the periodic table. 273 00:12:12,500 --> 00:12:13,510 Iron. 274 00:12:13,510 --> 00:12:15,320 Iron is everywhere. 275 00:12:15,320 --> 00:12:19,260 The most abundant element in terms of mass is iron. 276 00:12:19,260 --> 00:12:20,450 OK? 277 00:12:20,450 --> 00:12:25,610 And so, iron, you might expect from this description, 278 00:12:25,610 --> 00:12:26,570 to be front and center. 279 00:12:26,570 --> 00:12:30,650 And in fact, it is front and center. 280 00:12:30,650 --> 00:12:33,890 And so the other thing I think you can think about 281 00:12:33,890 --> 00:12:41,000 is solubilities and evolution of-- 282 00:12:41,000 --> 00:12:44,580 from the beginning, where we were in a completely anaerobic 283 00:12:44,580 --> 00:12:45,080 world. 284 00:12:45,080 --> 00:12:47,870 So here's the gaseous environment 285 00:12:47,870 --> 00:12:53,240 with oxygen. In the very beginning up to 2.4 286 00:12:53,240 --> 00:12:57,330 billion years ago, it was completely anaerobic. 287 00:12:57,330 --> 00:12:57,830 OK? 288 00:12:57,830 --> 00:12:59,790 And so is that important? 289 00:12:59,790 --> 00:13:06,440 So if we go to 2.4 billion, it's anaerobic. 290 00:13:06,440 --> 00:13:08,600 So now, if you look at-- 291 00:13:08,600 --> 00:13:11,113 and this is-- where these data come from 292 00:13:11,113 --> 00:13:13,530 and where this model comes from, it-- obviously everything 293 00:13:13,530 --> 00:13:14,510 is a model. 294 00:13:14,510 --> 00:13:16,430 You can go back and read this in detail 295 00:13:16,430 --> 00:13:18,350 if you become interested-- maybe some of you 296 00:13:18,350 --> 00:13:20,220 might have had a geology course where 297 00:13:20,220 --> 00:13:22,940 you've discussed this before. 298 00:13:22,940 --> 00:13:27,110 But if you look over here, where do you see iron? 299 00:13:27,110 --> 00:13:28,760 OK, so iron is going to be the focus. 300 00:13:28,760 --> 00:13:32,900 Where is it compared to cobalt, nickel, manganese, 301 00:13:32,900 --> 00:13:34,760 all these other-- zinc, copper-- 302 00:13:34,760 --> 00:13:37,160 all these other transition metals? 303 00:13:37,160 --> 00:13:38,180 It's way up here. 304 00:13:38,180 --> 00:13:42,440 So it's most abundant under anaerobic conditions. 305 00:13:42,440 --> 00:13:44,180 What do you think the oxidation state is? 306 00:13:48,450 --> 00:13:52,470 So you just told me you had iron that you've commonly encounter 307 00:13:52,470 --> 00:13:53,680 is two and three. 308 00:13:53,680 --> 00:13:54,925 And that's correct. 309 00:13:54,925 --> 00:13:57,300 Everything-- you're going to encounter this over and over 310 00:13:57,300 --> 00:13:57,800 again. 311 00:13:57,800 --> 00:14:00,570 Hopefully you have encountered this before. 312 00:14:00,570 --> 00:14:02,520 What happens in an anaerobic world? 313 00:14:02,520 --> 00:14:04,827 What do you think the oxidation state for iron is? 314 00:14:04,827 --> 00:14:05,660 AUDIENCE: Maybe two? 315 00:14:05,660 --> 00:14:06,493 JOANNE STUBBE: Yeah. 316 00:14:06,493 --> 00:14:07,770 It's two. 317 00:14:07,770 --> 00:14:12,780 And I think this is incredibly important from a chemical 318 00:14:12,780 --> 00:14:18,210 perspective, because many enzymes we're going to see-- 319 00:14:18,210 --> 00:14:22,320 metals-- can catalyze reactions by polarizing carbonyls, 320 00:14:22,320 --> 00:14:23,850 for example. 321 00:14:23,850 --> 00:14:26,280 In an anaerobic world, you likely 322 00:14:26,280 --> 00:14:28,770 used iron two all the time. 323 00:14:28,770 --> 00:14:31,290 But what's going to happen when we get over here 324 00:14:31,290 --> 00:14:33,750 in an aerobic world? 325 00:14:33,750 --> 00:14:35,790 And so that's the key question. 326 00:14:35,790 --> 00:14:38,910 And do we see iron two used in that capacity? 327 00:14:38,910 --> 00:14:42,060 The answer is no, because in the presence of oxygen 328 00:14:42,060 --> 00:14:44,200 some other reaction out competes it. 329 00:14:44,200 --> 00:14:46,133 So that's why I'm introducing you to this. 330 00:14:46,133 --> 00:14:48,550 It's sort of-- I don't expect you to remember the details, 331 00:14:48,550 --> 00:14:53,610 but I think it's an interesting exercise to think about what 332 00:14:53,610 --> 00:14:58,230 happened when we transitioned from an anaerobic world-- 333 00:14:58,230 --> 00:15:00,690 and this is all in the ocean, and versus the atmosphere-- 334 00:15:00,690 --> 00:15:04,060 into an oxygen atmosphere. 335 00:15:04,060 --> 00:15:08,790 And this is 0.8 billion years later. 336 00:15:08,790 --> 00:15:10,800 And if you look at this, what happens-- 337 00:15:10,800 --> 00:15:13,320 and this is a period where they believe that you 338 00:15:13,320 --> 00:15:16,440 had a lot of H2S around. 339 00:15:16,440 --> 00:15:19,340 And remember, we just saw iron clusters 340 00:15:19,340 --> 00:15:21,000 with all these sulfides on them. 341 00:15:21,000 --> 00:15:25,020 Iron sulfur was in the prebiotic world. 342 00:15:25,020 --> 00:15:26,580 They can self assemble. 343 00:15:26,580 --> 00:15:28,320 They do all this kind of chemistry 344 00:15:28,320 --> 00:15:31,680 that-- until they knew about this radical SAM super family-- 345 00:15:31,680 --> 00:15:34,980 they thought was one electron, oxidation, and reduction. 346 00:15:34,980 --> 00:15:37,140 And nothing could be farther from the truth. 347 00:15:37,140 --> 00:15:39,450 Iron sulfur clusters play a key role, for example, 348 00:15:39,450 --> 00:15:41,170 in DNA replication. 349 00:15:41,170 --> 00:15:44,370 OK, so I think-- 350 00:15:44,370 --> 00:15:47,430 thinking about this and where these iron sulfur clusters 351 00:15:47,430 --> 00:15:50,850 came from, you provided some insight perhaps 352 00:15:50,850 --> 00:15:55,710 from looking at the geological record of what 353 00:15:55,710 --> 00:15:58,150 people think was occurring. 354 00:15:58,150 --> 00:16:02,310 So we went through a period where you had a lot of H2S. 355 00:16:02,310 --> 00:16:06,090 Concentrations of species have changed. 356 00:16:06,090 --> 00:16:09,610 And then we move into the aerobic world, 357 00:16:09,610 --> 00:16:11,170 and what happens here? 358 00:16:11,170 --> 00:16:12,480 So what happens to the iron? 359 00:16:16,340 --> 00:16:18,530 It's dramatically decreased. 360 00:16:18,530 --> 00:16:24,920 So when we go from the anaerobic to the aerobic, 361 00:16:24,920 --> 00:16:27,530 why does the iron-- what happens to the oxidation 362 00:16:27,530 --> 00:16:30,330 state of the iron? 363 00:16:30,330 --> 00:16:32,550 It gets oxidized to iron three. 364 00:16:32,550 --> 00:16:37,620 So we're changing in the oxidation state, 365 00:16:37,620 --> 00:16:39,550 and so we're going to have to deal with it. 366 00:16:39,550 --> 00:16:41,175 So I'm going to show you, this presents 367 00:16:41,175 --> 00:16:45,900 a major issue we face now, both as humans and as bacteria. 368 00:16:45,900 --> 00:16:50,250 If you look at this, what happens to copper and zinc-- 369 00:16:50,250 --> 00:16:52,830 if you believe this model? 370 00:16:52,830 --> 00:16:58,570 That the copper and zinc concentrations increase. 371 00:16:58,570 --> 00:17:00,510 And in fact, that becomes really important. 372 00:17:00,510 --> 00:17:02,830 Because if you look at the biological record, 373 00:17:02,830 --> 00:17:05,819 and you look at archae and bacteria that 374 00:17:05,819 --> 00:17:07,980 are much much, much older, what you see 375 00:17:07,980 --> 00:17:11,040 is-- you don't see that many copper catalyzed reactions 376 00:17:11,040 --> 00:17:14,670 in zinc, which has a really important role in humans, 377 00:17:14,670 --> 00:17:15,960 with zinc fingers. 378 00:17:15,960 --> 00:17:20,730 Doesn't play a role like that in bacterial systems. 379 00:17:20,730 --> 00:17:25,079 So I think this represents an interesting way 380 00:17:25,079 --> 00:17:31,140 to think about metal speciation, oxidation states, what ligands 381 00:17:31,140 --> 00:17:34,050 are going to be involved in what's happening-- 382 00:17:34,050 --> 00:17:39,270 and also what's happening in bacterial systems. 383 00:17:39,270 --> 00:17:42,780 The key thing that I want you to remember about this 384 00:17:42,780 --> 00:17:45,885 is that in the aerobic world-- 385 00:17:48,420 --> 00:17:53,310 so we now go from iron two to iron three. 386 00:17:53,310 --> 00:17:56,040 And what we'll see is the solubility properties of iron 387 00:17:56,040 --> 00:17:58,120 three are dramatically different, 388 00:17:58,120 --> 00:18:01,380 and that's something we're going to have to deal with. 389 00:18:01,380 --> 00:18:02,628 How do we get-- 390 00:18:02,628 --> 00:18:04,920 we talked about this last time-- how do we get iron out 391 00:18:04,920 --> 00:18:06,510 of a rock? 392 00:18:06,510 --> 00:18:08,393 OK, so that's an issue if you're a bacteria-- 393 00:18:08,393 --> 00:18:09,560 you have to figure that out. 394 00:18:09,560 --> 00:18:12,510 And bacteria have done some pretty cool things 395 00:18:12,510 --> 00:18:14,470 to figure that out. 396 00:18:14,470 --> 00:18:21,010 And so-- OK, so this, I think, also has-- 397 00:18:21,010 --> 00:18:22,830 we're going to be focusing on iron here-- 398 00:18:22,830 --> 00:18:26,430 important implications in terms of the chemistry. 399 00:18:26,430 --> 00:18:32,710 So in terms of being in an anaerobic world, 400 00:18:32,710 --> 00:18:37,980 we can use iron as a Lewis acid. 401 00:18:37,980 --> 00:18:39,360 OK? 402 00:18:39,360 --> 00:18:41,220 And so it can polarize a carbonyl. 403 00:18:41,220 --> 00:18:44,550 We'll come back to this in a minute. 404 00:18:44,550 --> 00:18:48,120 Nowadays, we almost never use iron two 405 00:18:48,120 --> 00:18:51,000 as a Lewis acid in biological systems. 406 00:18:51,000 --> 00:18:52,600 And why is that true? 407 00:18:52,600 --> 00:18:56,130 Because when we transitioned to the aerobic world, 408 00:18:56,130 --> 00:19:00,522 now we have this problem of-- 409 00:19:00,522 --> 00:19:03,870 that the iron three is what? 410 00:19:03,870 --> 00:19:04,800 It's insoluble. 411 00:19:07,650 --> 00:19:09,270 So that's one problem. 412 00:19:09,270 --> 00:19:14,370 And the second problem is that since it's insoluble, 413 00:19:14,370 --> 00:19:15,600 we can't use it. 414 00:19:15,600 --> 00:19:18,180 How do you get-- how do you get it to actually use it 415 00:19:18,180 --> 00:19:18,840 for chemistry? 416 00:19:18,840 --> 00:19:20,840 We're going to-- we're going to talk about that. 417 00:19:20,840 --> 00:19:22,470 How do you get it to look at chemistry. 418 00:19:22,470 --> 00:19:29,770 And then we have this issue of oxidation with oxygen, 419 00:19:29,770 --> 00:19:35,070 and this is going to lead us into module seven. 420 00:19:35,070 --> 00:19:37,350 So while in the very beginning, we 421 00:19:37,350 --> 00:19:42,600 used iron to do a lot of chemistry without oxygen 422 00:19:42,600 --> 00:19:43,950 around. 423 00:19:43,950 --> 00:19:46,260 We then moved into an oxygen-- 424 00:19:46,260 --> 00:19:49,920 oxyphilic world, and we have this issue 425 00:19:49,920 --> 00:19:52,710 of during this oxidation using oxygen 426 00:19:52,710 --> 00:19:54,000 as the oxidant-- what happens? 427 00:19:54,000 --> 00:19:56,690 You produce reactive oxygen species. 428 00:19:56,690 --> 00:19:57,190 OK. 429 00:19:57,190 --> 00:20:00,800 So, and then you have also the problem of insolubility. 430 00:20:00,800 --> 00:20:03,270 So you generated-- by making this transition 431 00:20:03,270 --> 00:20:08,340 into an oxyphilic world you're encountering two major problems 432 00:20:08,340 --> 00:20:10,440 that we're focused on. 433 00:20:10,440 --> 00:20:12,600 How do you deal with the insolubility problem 434 00:20:12,600 --> 00:20:18,480 and how do you deal with reactive oxygen species? 435 00:20:18,480 --> 00:20:21,160 And that's going to be-- following this module, 436 00:20:21,160 --> 00:20:25,830 we're going to talk about what happens with reactive oxygen 437 00:20:25,830 --> 00:20:29,070 species as a consequence of moving from an anaerobic 438 00:20:29,070 --> 00:20:31,887 to an aerobic world. 439 00:20:31,887 --> 00:20:33,720 I don't want to spend a lot of time on this, 440 00:20:33,720 --> 00:20:36,127 but I want to make sure that you understand 441 00:20:36,127 --> 00:20:37,710 there are some kinds of reactions that 442 00:20:37,710 --> 00:20:40,350 are really distinct from the reactions you 443 00:20:40,350 --> 00:20:42,400 meet in the organic world. 444 00:20:42,400 --> 00:20:43,620 And a lot of you-- 445 00:20:43,620 --> 00:20:46,290 we looked at the vitamin bottle, we learn a lot about flavins, 446 00:20:46,290 --> 00:20:48,810 we learn about pyridoxine, we learn about vitamin C-- 447 00:20:48,810 --> 00:20:50,520 all the vitamins we learn about. 448 00:20:50,520 --> 00:20:55,590 But we sort of ignore the metals on our bottle 449 00:20:55,590 --> 00:20:56,890 that's required for life. 450 00:20:56,890 --> 00:21:00,600 And so I don't want to spend a lot of time, but there are-- 451 00:21:00,600 --> 00:21:02,080 what are the general reactions? 452 00:21:02,080 --> 00:21:04,680 So I just want to say a little bit about general reactions. 453 00:21:08,190 --> 00:21:09,360 OK. 454 00:21:09,360 --> 00:21:12,180 And one of them is this idea of Lewis acid-- 455 00:21:12,180 --> 00:21:15,630 or Bronsted acid. 456 00:21:15,630 --> 00:21:21,300 And so what you can have is a carbonyl, 457 00:21:21,300 --> 00:21:25,210 and you can have a metal that can activate the carbonyl you 458 00:21:25,210 --> 00:21:26,610 for nucleophilic attack. 459 00:21:26,610 --> 00:21:27,210 OK? 460 00:21:27,210 --> 00:21:30,120 Where have we seen this before? 461 00:21:30,120 --> 00:21:33,090 We've seen this before in-- if you go back 462 00:21:33,090 --> 00:21:38,040 and you look in the glycolysis pathway, 463 00:21:38,040 --> 00:21:41,360 lots of times you use zinc to activate the carbonyl. 464 00:21:41,360 --> 00:21:43,110 Sometimes you use shift spaces, maybe. 465 00:21:43,110 --> 00:21:44,380 You remember that? 466 00:21:44,380 --> 00:21:47,500 In aldehyde dehydrogenase, aldehyde oxidate 467 00:21:47,500 --> 00:21:49,695 that converts aldehyde to an acid or reduces 468 00:21:49,695 --> 00:21:51,630 aldehyde to an alcohol-- 469 00:21:51,630 --> 00:21:52,740 they use zinc. 470 00:21:52,740 --> 00:21:53,670 OK? 471 00:21:53,670 --> 00:21:58,080 In the completely anaerobic world, people thought-- 472 00:21:58,080 --> 00:22:00,000 most of the time they probably used iron. 473 00:22:00,000 --> 00:22:03,060 That was one of the most-- that was much, much more prevalent 474 00:22:03,060 --> 00:22:04,440 than zinc. 475 00:22:04,440 --> 00:22:06,660 But then things-- so if you go way back 476 00:22:06,660 --> 00:22:09,840 and you find bacteria that lived in that period, 477 00:22:09,840 --> 00:22:12,210 they still might be using iron in catalysis. 478 00:22:12,210 --> 00:22:16,020 But now we almost never use iron two in catalysis, 479 00:22:16,020 --> 00:22:19,680 because of the issue of the redox chemistry. 480 00:22:19,680 --> 00:22:21,000 So now they're saying the-- 481 00:22:21,000 --> 00:22:23,940 so it's now, you know, your polarize 482 00:22:23,940 --> 00:22:25,740 this for a nucleophilic attack. 483 00:22:25,740 --> 00:22:27,720 You've seen this over and over again 484 00:22:27,720 --> 00:22:31,350 with the Claisen reaction, the Aldol reactions, et cetera. 485 00:22:31,350 --> 00:22:33,150 I'm not going to go through the details. 486 00:22:33,150 --> 00:22:35,760 Another place you see it-- 487 00:22:35,760 --> 00:22:38,760 and where have we seen this one? 488 00:22:38,760 --> 00:22:41,640 Again we have a metal-- 489 00:22:41,640 --> 00:22:44,370 and I'll just leave it in the plus two oxidation state. 490 00:22:44,370 --> 00:22:52,200 But what happens to the pKa of the water bound to a metal? 491 00:22:52,200 --> 00:22:58,530 And what happens is the pKa is dramatically reduced. 492 00:22:58,530 --> 00:23:00,150 You have two positive charges here, 493 00:23:00,150 --> 00:23:03,390 depending on the interact-- and that interaction's unfavorable. 494 00:23:03,390 --> 00:23:06,300 So the pKa becomes reduced on bonding to a metal. 495 00:23:06,300 --> 00:23:08,160 Where we've seen that before? 496 00:23:08,160 --> 00:23:09,810 We saw that in the cholesterol module. 497 00:23:09,810 --> 00:23:12,240 We didn't talk about the chemistry-- again, 498 00:23:12,240 --> 00:23:13,860 I come from the chemistry side of it 499 00:23:13,860 --> 00:23:16,290 so I find the chemistry the most interesting-- but it 500 00:23:16,290 --> 00:23:18,098 fits into the biology. 501 00:23:18,098 --> 00:23:19,390 Where have we seen this before? 502 00:23:19,390 --> 00:23:21,180 Anybody remember-- in cholesterol? 503 00:23:21,180 --> 00:23:24,250 Homeostasis? 504 00:23:24,250 --> 00:23:27,490 What happens in the Golgi when you want to go from the Golgi 505 00:23:27,490 --> 00:23:28,960 to the nucleus? 506 00:23:28,960 --> 00:23:29,960 AUDIENCE: A zinc-- 507 00:23:29,960 --> 00:23:31,793 JOANNE STUBBE: Yeah, we had a zinc protease. 508 00:23:31,793 --> 00:23:32,980 So that would be an example. 509 00:23:32,980 --> 00:23:38,920 An example of this would be in the cholesterol section. 510 00:23:38,920 --> 00:23:41,650 And I'm not going to talk about this in detail. 511 00:23:41,650 --> 00:23:43,820 I used to talk about this in a lot more detail, 512 00:23:43,820 --> 00:23:45,820 but you can see with different metals-- 513 00:23:45,820 --> 00:23:49,420 this is just an example of the first case I'm giving you-- 514 00:23:49,420 --> 00:23:54,840 the pKa's of the metal bound are reduced. 515 00:23:54,840 --> 00:23:56,830 Again, it's a play off. 516 00:23:56,830 --> 00:23:58,942 Those all with waters, those ligands. 517 00:23:58,942 --> 00:24:00,650 Every time you start changing the ligands 518 00:24:00,650 --> 00:24:03,250 or you change the oxidation state, these numbers change. 519 00:24:03,250 --> 00:24:03,760 OK? 520 00:24:03,760 --> 00:24:08,290 So you need to know a lot about the metal you're dealing with. 521 00:24:08,290 --> 00:24:10,810 So that's one place-- you've already seen all of this 522 00:24:10,810 --> 00:24:13,020 before. 523 00:24:13,020 --> 00:24:14,950 Whoops. 524 00:24:14,950 --> 00:24:17,860 So the second thing I want to very briefly talk about 525 00:24:17,860 --> 00:24:22,450 is the second kind of reaction-- which maybe many of you 526 00:24:22,450 --> 00:24:24,885 haven't seen before-- is electron transfer. 527 00:24:30,460 --> 00:24:30,990 OK. 528 00:24:30,990 --> 00:24:36,670 So this is basically oxidation reduction. 529 00:24:36,670 --> 00:24:41,470 And so clearly, you've seen oxidation reduction. 530 00:24:41,470 --> 00:24:45,930 So if we have some metal m in the n plus state, 531 00:24:45,930 --> 00:24:48,870 and we add an electron, it gets reduced. 532 00:24:52,020 --> 00:24:54,450 So to get to the reduced state, remember 533 00:24:54,450 --> 00:24:56,630 we need two half reactions-- something gets reduced, 534 00:24:56,630 --> 00:25:00,180 something else has to get oxidized. 535 00:25:00,180 --> 00:25:03,960 And what's different-- we've looked at redox cofactors-- 536 00:25:03,960 --> 00:25:07,290 and most of you have looked at a lot of redox cofactors 537 00:25:07,290 --> 00:25:09,950 in primary metabolism, like glycolysis 538 00:25:09,950 --> 00:25:12,240 of the pentose phosphate pathway or whatever-- 539 00:25:12,240 --> 00:25:15,570 what are the normal redox cofactors you encounter? 540 00:25:15,570 --> 00:25:18,315 The organic redox cofactors you encounter in biology? 541 00:25:21,312 --> 00:25:22,210 AUDIENCE: NAD. 542 00:25:22,210 --> 00:25:24,250 JOANNE STUBBE: Yeah, NAD-- 543 00:25:24,250 --> 00:25:25,430 NAD flavins. 544 00:25:25,430 --> 00:25:30,760 OK, so this chemistry always involves one electron. 545 00:25:30,760 --> 00:25:32,300 So that's distinct. 546 00:25:32,300 --> 00:25:34,730 NAD, we've already talked about this, always involves 547 00:25:34,730 --> 00:25:36,500 hydride transfer-- 548 00:25:36,500 --> 00:25:38,440 two electrons and a proton. 549 00:25:38,440 --> 00:25:41,450 So this is one electron. 550 00:25:41,450 --> 00:25:42,950 OK. 551 00:25:42,950 --> 00:25:45,470 And so one electron. 552 00:25:45,470 --> 00:25:47,630 And if you have other things-- 553 00:25:47,630 --> 00:25:53,030 we could have proton coupled electron transfer. 554 00:25:53,030 --> 00:25:59,480 So PC is proton coupled electron transfer. 555 00:25:59,480 --> 00:26:01,400 And remember, we just saw the example 556 00:26:01,400 --> 00:26:04,430 of nitrogen getting reduced to ammonia. 557 00:26:04,430 --> 00:26:05,300 OK? 558 00:26:05,300 --> 00:26:07,550 You're doing an eight-electron reduction, 559 00:26:07,550 --> 00:26:09,590 but you've got to have protons. 560 00:26:09,590 --> 00:26:12,800 That involves proton coupled electron transfer. 561 00:26:12,800 --> 00:26:16,090 If you're converting water into oxygen, 562 00:26:16,090 --> 00:26:19,160 again, you've got to take care of the electrons 563 00:26:19,160 --> 00:26:20,120 and the protons. 564 00:26:20,120 --> 00:26:24,080 And if I get that far in the last module, 565 00:26:24,080 --> 00:26:26,510 my lab works on ribonucelotide reductases-- 566 00:26:26,510 --> 00:26:29,330 that makes a precursor to DNA. 567 00:26:29,330 --> 00:26:31,790 You would never think about radicals, at all, 568 00:26:31,790 --> 00:26:34,280 but that chemistry involves proton 569 00:26:34,280 --> 00:26:35,900 coupled electron transfer. 570 00:26:35,900 --> 00:26:38,975 So here is some of the most important reactions in biology, 571 00:26:38,975 --> 00:26:40,850 and you really haven't been exposed to what's 572 00:26:40,850 --> 00:26:43,610 unique about the chemistry. 573 00:26:43,610 --> 00:26:46,130 So what do we know that's unique about the chemistry? 574 00:26:46,130 --> 00:26:50,630 What do what do we know about rate constants for electron 575 00:26:50,630 --> 00:26:51,800 transfer? 576 00:26:51,800 --> 00:26:52,850 Anybody know anything? 577 00:26:56,300 --> 00:26:57,720 Fast, slow. 578 00:26:57,720 --> 00:27:00,575 What's different about electron versus hydride transfer? 579 00:27:04,130 --> 00:27:05,630 AUDIENCE: With the hydride transfer, 580 00:27:05,630 --> 00:27:08,030 you have to transfer an entire proton, versus-- 581 00:27:08,030 --> 00:27:10,355 JOANNE STUBBE: So you're transferring the proton, 582 00:27:10,355 --> 00:27:12,730 which-- what's the difference in mass between an electron 583 00:27:12,730 --> 00:27:13,310 and a proton? 584 00:27:13,310 --> 00:27:14,220 AUDIENCE: A lot. 585 00:27:14,220 --> 00:27:15,053 JOANNE STUBBE: Huge. 586 00:27:15,053 --> 00:27:17,110 It's 2,000-- 2,000 fold. 587 00:27:17,110 --> 00:27:20,110 So you remember, probably from introductory chemistry, 588 00:27:20,110 --> 00:27:23,550 when you think about electrons, you think about-- 589 00:27:23,550 --> 00:27:26,080 you think about quantum mechanics and quantum 590 00:27:26,080 --> 00:27:29,440 tunneling, as well as-- it can be-- 591 00:27:29,440 --> 00:27:32,110 electrons can function as both particles and waves. 592 00:27:32,110 --> 00:27:37,900 So they can function as waves and particles. 593 00:27:37,900 --> 00:27:40,150 And while I'm not going to spend a lot of time talking 594 00:27:40,150 --> 00:27:42,940 about this, this is a central reaction 595 00:27:42,940 --> 00:27:45,880 in the inorganic part of biochemistry 596 00:27:45,880 --> 00:27:49,900 that occurs in humans that you need to take into account. 597 00:27:49,900 --> 00:27:51,940 When things behave as waves, they 598 00:27:51,940 --> 00:27:54,230 can function quantum mechanically. 599 00:27:54,230 --> 00:27:57,460 And we have an expression called the Marcus equation, 600 00:27:57,460 --> 00:28:00,250 which allows us to calculate the rate constants. 601 00:28:00,250 --> 00:28:04,540 So we have a rate constant for electron transfer. 602 00:28:04,540 --> 00:28:07,960 And if we have some acceptor her and some donor-- so, 603 00:28:07,960 --> 00:28:09,760 all we're doing is redox chemistry. 604 00:28:13,420 --> 00:28:15,640 The question is, what governs the rate constants 605 00:28:15,640 --> 00:28:16,840 for electron transfer? 606 00:28:16,840 --> 00:28:21,070 Well, it could be the electronic overlap, so that's part of it. 607 00:28:21,070 --> 00:28:22,930 This is part of the Marcus equation. 608 00:28:25,900 --> 00:28:28,330 What else governs the-- 609 00:28:28,330 --> 00:28:30,100 what else governs the redox chemistry 610 00:28:30,100 --> 00:28:32,160 if you have a donor and acceptor? 611 00:28:32,160 --> 00:28:34,960 The reduction potential of the donor and acceptor. 612 00:28:34,960 --> 00:28:39,370 So you need to think about the reduction potentials. 613 00:28:42,040 --> 00:28:46,150 And what other factor governs the chemistry? 614 00:28:46,150 --> 00:28:47,080 Does anybody know? 615 00:28:47,080 --> 00:28:47,860 Around the metals. 616 00:28:47,860 --> 00:28:50,890 So you have to think about, how much energy 617 00:28:50,890 --> 00:28:53,500 does it take to go from iron two to iron three, 618 00:28:53,500 --> 00:28:56,050 or copper two to copper one? 619 00:28:56,050 --> 00:28:57,160 What other factor? 620 00:28:57,160 --> 00:29:00,850 What else happens to the metal during a reduction 621 00:29:00,850 --> 00:29:02,292 or an oxidation? 622 00:29:02,292 --> 00:29:03,470 AUDIENCE: A reorganization. 623 00:29:03,470 --> 00:29:05,012 JOANNE STUBBE: Yes, a reorganization. 624 00:29:05,012 --> 00:29:08,290 So it can change its geometry. 625 00:29:08,290 --> 00:29:10,960 And so the other factor is called lambda, 626 00:29:10,960 --> 00:29:18,480 and this is reorganization chemistry. 627 00:29:18,480 --> 00:29:21,900 And furthermore-- and we'll see this is important a little bit 628 00:29:21,900 --> 00:29:23,820 with the iron systems-- 629 00:29:23,820 --> 00:29:26,490 it doesn't just have to be the immediate coordination 630 00:29:26,490 --> 00:29:28,140 sphere of the metals. 631 00:29:28,140 --> 00:29:31,170 It can be the second coordination sphere, as well. 632 00:29:31,170 --> 00:29:33,090 So the whole protein is important, 633 00:29:33,090 --> 00:29:36,090 I think as hopefully most of you know by now. 634 00:29:36,090 --> 00:29:38,430 And we're not going to spend a lot of time on this, 635 00:29:38,430 --> 00:29:40,260 but I think this is something you 636 00:29:40,260 --> 00:29:43,620 need to think about-- the rate constants for electron 637 00:29:43,620 --> 00:29:44,310 transfer. 638 00:29:44,310 --> 00:29:47,790 They could be 10 to the eighth, 10 to the 10th per second. 639 00:29:47,790 --> 00:29:50,640 How does that compare with the rate constant for chymotrypsin? 640 00:29:56,840 --> 00:29:58,590 What's the turnover number for a protease? 641 00:30:01,480 --> 00:30:04,240 Anybody remember? 642 00:30:04,240 --> 00:30:04,740 OK. 643 00:30:04,740 --> 00:30:07,980 So a turnover number for a typical protease hydrolyzes-- 644 00:30:07,980 --> 00:30:11,490 like the cholesterol one hydrolyzes on an amine bond-- 645 00:30:11,490 --> 00:30:14,620 might be anywhere from 10 to 50 per second. 646 00:30:14,620 --> 00:30:15,360 OK. 647 00:30:15,360 --> 00:30:17,790 So how does that compare to this? 648 00:30:17,790 --> 00:30:18,840 Slow. 649 00:30:18,840 --> 00:30:20,040 Very slow. 650 00:30:20,040 --> 00:30:22,200 So again, the chemistry of electron transfer 651 00:30:22,200 --> 00:30:26,010 is quite distinct from most of the chemistry 652 00:30:26,010 --> 00:30:27,750 you've encountered, and so you need 653 00:30:27,750 --> 00:30:30,150 to know it exists because it's everywhere in biology. 654 00:30:30,150 --> 00:30:33,060 We don't spend that much time on it in this class, 655 00:30:33,060 --> 00:30:38,460 but it's a unique part of the chemistry associated 656 00:30:38,460 --> 00:30:38,995 with metals. 657 00:30:41,650 --> 00:30:44,250 OK, so the third thing I wanted-- the third kind 658 00:30:44,250 --> 00:30:47,540 of chemistry I want to very briefly look at 659 00:30:47,540 --> 00:30:49,320 is substitution reactions. 660 00:30:54,570 --> 00:30:55,500 OK. 661 00:30:55,500 --> 00:30:59,520 Now, in organic chemistry, what kind of substitutions reactions 662 00:30:59,520 --> 00:31:01,215 do you have? 663 00:31:01,215 --> 00:31:03,840 This is something hopefully you all remember from your organic, 664 00:31:03,840 --> 00:31:05,298 but what do you-- what do you have? 665 00:31:05,298 --> 00:31:07,530 What are the two basic reactions you 666 00:31:07,530 --> 00:31:11,496 learn about in the first semester of organic chemistry? 667 00:31:11,496 --> 00:31:12,870 AUDIENCE: SN1 and SN2. 668 00:31:12,870 --> 00:31:13,745 JOANNE STUBBE: Right. 669 00:31:13,745 --> 00:31:14,270 SN1, SN2. 670 00:31:14,270 --> 00:31:15,890 Associate or dissociate. 671 00:31:15,890 --> 00:31:17,930 Same thing in metals, OK? 672 00:31:17,930 --> 00:31:21,740 So you need to think about associative-- 673 00:31:21,740 --> 00:31:23,360 what does that mean? 674 00:31:23,360 --> 00:31:25,970 Dissociative. 675 00:31:25,970 --> 00:31:27,525 If you have something with four-- 676 00:31:27,525 --> 00:31:30,170 a metal with four ligands around it, 677 00:31:30,170 --> 00:31:32,840 you're going to add a ligand to get the reaction to go. 678 00:31:32,840 --> 00:31:33,920 That's associative. 679 00:31:33,920 --> 00:31:36,025 If you have something with four ligands around it, 680 00:31:36,025 --> 00:31:37,525 one of the ligands could dissociate, 681 00:31:37,525 --> 00:31:38,900 and you only have three ligands-- 682 00:31:38,900 --> 00:31:42,650 and that's the basis for getting that chemistry to go. 683 00:31:42,650 --> 00:31:47,210 And the reason-- the thing that I want to focus on and-- 684 00:31:47,210 --> 00:31:49,925 the thing I want to focus on is ligand exchange. 685 00:31:52,460 --> 00:31:58,640 So ligand exchange could occur by associative or dissociative 686 00:31:58,640 --> 00:31:59,390 mechanisms. 687 00:31:59,390 --> 00:32:01,850 Where have you seen ligand exchange in recitation? 688 00:32:04,520 --> 00:32:05,960 I think it was recitation four? 689 00:32:11,572 --> 00:32:13,030 You probably didn't think about it. 690 00:32:13,030 --> 00:32:14,572 I mean, we were doing something else. 691 00:32:14,572 --> 00:32:20,500 But the key to it working is ligand exchange rates. 692 00:32:20,500 --> 00:32:23,770 What about histamine tags? 693 00:32:23,770 --> 00:32:25,600 OK, so here you have a metal. 694 00:32:25,600 --> 00:32:27,760 What kind of a metal do you have on your column? 695 00:32:27,760 --> 00:32:28,930 A nickel. 696 00:32:28,930 --> 00:32:30,490 And the nickel is bound. 697 00:32:30,490 --> 00:32:32,740 But in order-- so you can hang-- 698 00:32:32,740 --> 00:32:34,090 how does your thing hang up? 699 00:32:34,090 --> 00:32:35,290 By ligand exchange. 700 00:32:35,290 --> 00:32:36,300 How does it come off? 701 00:32:36,300 --> 00:32:37,780 By ligand exchange. 702 00:32:37,780 --> 00:32:41,670 So an example of this is histamine tag chemistry. 703 00:32:45,020 --> 00:32:49,990 And another example that you've seen is magnesium. 704 00:32:49,990 --> 00:32:51,770 OK. 705 00:32:51,770 --> 00:32:54,670 What are the rate constants for ligand exchange with magnesium? 706 00:32:54,670 --> 00:32:58,032 Where do we see magnesium in biology? 707 00:32:58,032 --> 00:32:59,490 I'm spending too much time on this. 708 00:32:59,490 --> 00:33:01,573 But I actually think this is incredibly important. 709 00:33:01,573 --> 00:33:04,592 If you take home a few of these basic reactions, 710 00:33:04,592 --> 00:33:06,050 this is all you really sort of need 711 00:33:06,050 --> 00:33:11,130 to know to deal with metals and biological systems. 712 00:33:11,130 --> 00:33:12,405 Where's magnesium? 713 00:33:12,405 --> 00:33:13,280 Where do you find it? 714 00:33:13,280 --> 00:33:14,165 You find it on-- 715 00:33:14,165 --> 00:33:15,040 AUDIENCE: Phosphates. 716 00:33:15,040 --> 00:33:16,498 JOANNE STUBBE: On phosphates, yeah. 717 00:33:16,498 --> 00:33:21,500 So you have nucleotides-- like ATP would be an example. 718 00:33:21,500 --> 00:33:25,190 Whenever you have ATP, if you look at the charges of ATP-- 719 00:33:25,190 --> 00:33:28,400 we went through this in one of the recitations that I taught-- 720 00:33:28,400 --> 00:33:30,020 you never have these negative charges. 721 00:33:30,020 --> 00:33:31,190 It's always complex. 722 00:33:31,190 --> 00:33:32,990 Just something to neutralize it. 723 00:33:32,990 --> 00:33:35,300 And the major thing-- since magnesium is 10, 724 00:33:35,300 --> 00:33:38,450 15 millimolar inside the cell-- 725 00:33:38,450 --> 00:33:39,590 it's always bound. 726 00:33:39,590 --> 00:33:42,500 But if you try to isolate magnesium 727 00:33:42,500 --> 00:33:45,530 through some kind of a column, what happens? 728 00:33:45,530 --> 00:33:50,930 The magnesium-- because of the rate constants for exchange-- 729 00:33:50,930 --> 00:33:51,800 falls off. 730 00:33:51,800 --> 00:33:53,510 So if you have something else in there 731 00:33:53,510 --> 00:33:56,010 that can out compete it-- like protons or something-- 732 00:33:56,010 --> 00:33:56,510 it's gone. 733 00:33:56,510 --> 00:33:58,580 You never look at-- it depends on the rate 734 00:33:58,580 --> 00:34:00,500 constants for exchange-- but you never 735 00:34:00,500 --> 00:34:05,690 see the metal bound to those small molecules. 736 00:34:05,690 --> 00:34:07,695 So this is rapid exchange. 737 00:34:11,239 --> 00:34:19,764 And we'll see in the case of iron rapid exchange-- 738 00:34:19,764 --> 00:34:21,969 and I'm going to show you a table with this-- 739 00:34:21,969 --> 00:34:24,389 but rapid exchange is also important. 740 00:34:24,389 --> 00:34:26,389 And why is that important? 741 00:34:26,389 --> 00:34:29,800 It's important because say you isolate a protein 742 00:34:29,800 --> 00:34:31,690 and you're putting it through a column. 743 00:34:31,690 --> 00:34:35,050 What-- if the ligands are coming off and on, 744 00:34:35,050 --> 00:34:37,090 what happens to the metal by the time you get it 745 00:34:37,090 --> 00:34:38,440 out the bottom of the column? 746 00:34:38,440 --> 00:34:40,270 There's no metal. 747 00:34:40,270 --> 00:34:43,449 So the issues with iron, which is everywhere, 748 00:34:43,449 --> 00:34:47,949 that catalyzes many, many, many kinds of reactions, is it's 749 00:34:47,949 --> 00:34:50,380 really hard to tell that there was 750 00:34:50,380 --> 00:34:57,220 a metal there inside the cell, because the iron dissociates 751 00:34:57,220 --> 00:34:57,860 during-- 752 00:34:57,860 --> 00:34:59,890 in the plus two state-- 753 00:34:59,890 --> 00:35:03,940 during protein purification. 754 00:35:08,260 --> 00:35:10,750 So what about-- what if I changed the oxidation 755 00:35:10,750 --> 00:35:12,260 from iron two to iron three? 756 00:35:12,260 --> 00:35:14,974 What do you think would happen to the exchange rate? 757 00:35:14,974 --> 00:35:16,270 AUDIENCE: Slow down a lot. 758 00:35:16,270 --> 00:35:17,103 JOANNE STUBBE: Yeah. 759 00:35:17,103 --> 00:35:18,400 So it would slow down a lot. 760 00:35:18,400 --> 00:35:21,070 Every metal-- every metal is different. 761 00:35:21,070 --> 00:35:23,050 Every set of ligands is different. 762 00:35:23,050 --> 00:35:25,600 But you need to think about exchange reactions, 763 00:35:25,600 --> 00:35:28,990 because they're all over the place in biology. 764 00:35:28,990 --> 00:35:32,500 Here's is an example that I took out of Lippard's book. 765 00:35:32,500 --> 00:35:35,050 I used to give a lot more data than this, 766 00:35:35,050 --> 00:35:36,760 but these give you the rate constants 767 00:35:36,760 --> 00:35:38,530 for exchange for iron. 768 00:35:38,530 --> 00:35:40,750 Here you can see iron two, iron three-- and these 769 00:35:40,750 --> 00:35:41,980 are all waters. 770 00:35:41,980 --> 00:35:43,162 OK? 771 00:35:43,162 --> 00:35:45,370 That you're never going to sight see inside the cell. 772 00:35:45,370 --> 00:35:47,995 You might have a few waters, but you have other ligands around. 773 00:35:47,995 --> 00:35:51,520 All of the exchange rates change with different ligands, 774 00:35:51,520 --> 00:35:53,200 so you need to think about that. 775 00:35:53,200 --> 00:35:56,210 And also magnesium-- 6 times 10 to the fifth per second. 776 00:35:56,210 --> 00:35:58,210 So it's exchanging really rapidly. 777 00:35:58,210 --> 00:36:00,540 And that really does govern-- 778 00:36:00,540 --> 00:36:03,820 you know, here we're doing protein purification here. 779 00:36:03,820 --> 00:36:06,220 We're trying to identify what the metal is. 780 00:36:06,220 --> 00:36:08,020 This is it made it really challenging 781 00:36:08,020 --> 00:36:13,490 to tell whether you ever had iron two bound to your protein. 782 00:36:13,490 --> 00:36:16,750 Sometimes you isolate zinc bound to your protein. 783 00:36:16,750 --> 00:36:19,390 And I'm going to show you-- because of the periodic table, 784 00:36:19,390 --> 00:36:22,130 zinc always out competes iron. 785 00:36:22,130 --> 00:36:23,890 So when you're purifying something 786 00:36:23,890 --> 00:36:25,930 and you have zinc contaminant in your buffers 787 00:36:25,930 --> 00:36:29,800 and stuff like that, you'll get the iron replaced with zinc 788 00:36:29,800 --> 00:36:32,140 and think you have a zinc protein. 789 00:36:32,140 --> 00:36:32,770 And you don't. 790 00:36:32,770 --> 00:36:36,010 You really had an iron protein, but because of ligand exchange, 791 00:36:36,010 --> 00:36:39,580 you don't know what the real active form of the protein is. 792 00:36:39,580 --> 00:36:43,270 This is something that's plagued this area for a long time, 793 00:36:43,270 --> 00:36:48,520 and it certainly plagues the area of the iron 794 00:36:48,520 --> 00:36:50,710 that we're going to be focused on. 795 00:36:50,710 --> 00:36:51,490 So let me see. 796 00:36:51,490 --> 00:36:53,060 I think I want to go up one more. 797 00:36:53,060 --> 00:36:53,560 All right. 798 00:36:56,750 --> 00:36:58,640 What do I want to say now? 799 00:36:58,640 --> 00:37:02,960 So the other thing I want to talk about is-- 800 00:37:02,960 --> 00:37:07,880 that's unique and distinct from what you see in solution-- 801 00:37:07,880 --> 00:37:09,753 all of this stuff happens in solution. 802 00:37:09,753 --> 00:37:10,670 That's where we learn. 803 00:37:10,670 --> 00:37:13,040 Just like with organic cofactors. 804 00:37:13,040 --> 00:37:15,260 We sort of study them, we learn how they work, 805 00:37:15,260 --> 00:37:17,030 then we take them into biological systems. 806 00:37:17,030 --> 00:37:18,980 We use that as a starting point for 807 00:37:18,980 --> 00:37:21,050 think about-- thinking about how the enzymes use 808 00:37:21,050 --> 00:37:22,245 these cofactors. 809 00:37:22,245 --> 00:37:23,870 And in fact, what you learned over here 810 00:37:23,870 --> 00:37:26,120 is exactly what you learn over here, 811 00:37:26,120 --> 00:37:27,890 except nature has figured out how 812 00:37:27,890 --> 00:37:29,930 to catalyze the reactions by a factor of 10 813 00:37:29,930 --> 00:37:31,160 to the 12th faster. 814 00:37:31,160 --> 00:37:31,940 OK? 815 00:37:31,940 --> 00:37:35,120 So nature adds her-- 816 00:37:35,120 --> 00:37:37,880 adds her two cents worth on top of all 817 00:37:37,880 --> 00:37:40,260 the organic and inorganic chemistry we learn. 818 00:37:40,260 --> 00:37:43,130 And what is it that at this information? 819 00:37:43,130 --> 00:37:44,870 It's the protein environment. 820 00:37:44,870 --> 00:37:48,650 So the last thing that one really needs to think about 821 00:37:48,650 --> 00:37:58,340 is how do proteins tune metal properties? 822 00:38:04,720 --> 00:38:05,220 OK. 823 00:38:05,220 --> 00:38:06,780 So that's the big question. 824 00:38:06,780 --> 00:38:09,630 And we're going to spend a little bit of time 825 00:38:09,630 --> 00:38:10,590 talking about that. 826 00:38:10,590 --> 00:38:16,170 And to do that, I want to go back to the periodic table. 827 00:38:16,170 --> 00:38:22,000 OK, so again we're going to be focused on these metals. 828 00:38:22,000 --> 00:38:24,820 And what we see is there is a set 829 00:38:24,820 --> 00:38:28,210 of rules that inorganic chemists Irving and Williams-- 830 00:38:28,210 --> 00:38:32,020 many of you may have heard of the Irving-Williams series-- 831 00:38:32,020 --> 00:38:33,790 it sort of makes a prediction based 832 00:38:33,790 --> 00:38:36,910 on what you learned about transition metals in terms 833 00:38:36,910 --> 00:38:38,230 of ability to bind. 834 00:38:38,230 --> 00:38:41,890 If you compare all of these metals in the same oxidation 835 00:38:41,890 --> 00:38:45,890 state, in the same geometric environment. 836 00:38:45,890 --> 00:38:51,100 So one of the questions that we face is binding. 837 00:38:51,100 --> 00:38:52,430 And why is that important? 838 00:38:52,430 --> 00:38:58,630 Because inside the cell, we will see that copper binds much more 839 00:38:58,630 --> 00:39:01,330 tightly than manganese-- no matter what you do, 840 00:39:01,330 --> 00:39:02,350 that's true. 841 00:39:02,350 --> 00:39:03,730 And what's the basis of that? 842 00:39:03,730 --> 00:39:07,630 It's the atomic number, which changes the atomic radius-- 843 00:39:07,630 --> 00:39:08,760 it makes it smaller. 844 00:39:08,760 --> 00:39:12,710 It makes the ligands bind more tightly. 845 00:39:12,710 --> 00:39:14,800 So the problem is, when you're inside the cell-- 846 00:39:14,800 --> 00:39:17,200 if all these things were floating around 847 00:39:17,200 --> 00:39:19,960 inside the cell-- how do you control the metallation 848 00:39:19,960 --> 00:39:21,973 state inside the cell? 849 00:39:21,973 --> 00:39:24,140 So that's the key issue, and I'm going to give you-- 850 00:39:24,140 --> 00:39:25,973 I'm going to show you a little bit about how 851 00:39:25,973 --> 00:39:30,460 nature has figured out how to control all of this. 852 00:39:30,460 --> 00:39:34,300 It goes awry quite frequently, and that's-- 853 00:39:34,300 --> 00:39:35,720 how does it manifest itself? 854 00:39:35,720 --> 00:39:37,840 It manifests itself in disease. 855 00:39:37,840 --> 00:39:42,220 Just like we saw with cholesterol. 856 00:39:42,220 --> 00:39:43,840 So what we're going to see-- 857 00:39:43,840 --> 00:39:48,820 we are going to look at first row transition metals. 858 00:39:54,110 --> 00:39:56,330 In general, we'll see that manganese 859 00:39:56,330 --> 00:39:59,092 two binds less tightly-- 860 00:39:59,092 --> 00:40:00,800 if you look over there, you can see where 861 00:40:00,800 --> 00:40:03,180 we are in the periodic table. 862 00:40:03,180 --> 00:40:10,070 The atomic numbers increase less than nickel, less than copper. 863 00:40:10,070 --> 00:40:14,540 So here are-- here are our transition metals. 864 00:40:14,540 --> 00:40:22,050 And so what we see is the atomic numbers decrease-- 865 00:40:22,050 --> 00:40:22,550 increase. 866 00:40:26,340 --> 00:40:34,760 And the atomic radius decreases. 867 00:40:34,760 --> 00:40:37,890 And therefore what you see is over at this end, 868 00:40:37,890 --> 00:40:38,960 you have weak binding-- 869 00:40:38,960 --> 00:40:41,630 over at the manganese and iron end, we have weak binding. 870 00:40:47,810 --> 00:40:50,150 And over at this end, we have strong binding. 871 00:40:54,960 --> 00:40:56,300 So if you had a protein-- 872 00:40:56,300 --> 00:40:59,100 and I'm going to give you an example of this. 873 00:40:59,100 --> 00:41:00,850 There is a protein I'm going to show you 874 00:41:00,850 --> 00:41:03,560 that combined both copper and manganese. 875 00:41:03,560 --> 00:41:04,730 And you had equal amounts? 876 00:41:04,730 --> 00:41:06,200 Copper would always win-- 877 00:41:06,200 --> 00:41:07,250 by a lot. 878 00:41:07,250 --> 00:41:07,880 OK? 879 00:41:07,880 --> 00:41:10,250 So you need to study this, but you know-- 880 00:41:10,250 --> 00:41:13,700 you'd have to use 10,000 times more manganese to out compete 881 00:41:13,700 --> 00:41:14,690 the copper. 882 00:41:14,690 --> 00:41:15,200 OK? 883 00:41:15,200 --> 00:41:16,610 So this just shows you. 884 00:41:16,610 --> 00:41:21,860 So this is called the Irving-Williams series 885 00:41:21,860 --> 00:41:28,790 after the people who described this. 886 00:41:28,790 --> 00:41:31,598 And what they compared to get these numbers-- 887 00:41:31,598 --> 00:41:33,890 they are looking at all of these things in the plus two 888 00:41:33,890 --> 00:41:34,770 oxidation state. 889 00:41:34,770 --> 00:41:35,270 OK? 890 00:41:35,270 --> 00:41:37,760 And they're looking at it all in an octahedral environment, 891 00:41:37,760 --> 00:41:40,340 with six ligands around it. 892 00:41:40,340 --> 00:41:51,140 This is all plus two oxidation state, and all octahedral. 893 00:41:51,140 --> 00:41:54,950 Everybody remember octahedral? 894 00:41:54,950 --> 00:41:58,670 We have four equatorial ligands, and two axial ligands-- 895 00:41:58,670 --> 00:42:00,870 I'm not going to draw that out on the board. 896 00:42:00,870 --> 00:42:01,370 OK. 897 00:42:01,370 --> 00:42:03,500 So that's an issue. 898 00:42:03,500 --> 00:42:08,450 And the question then is, how do we deal with this issue? 899 00:42:08,450 --> 00:42:11,150 So here's our Irving-Williams series 900 00:42:11,150 --> 00:42:12,230 that I've given you here. 901 00:42:12,230 --> 00:42:14,212 But what do we do-- 902 00:42:14,212 --> 00:42:15,920 how do we deal with this inside the cell? 903 00:42:20,970 --> 00:42:25,350 The issue is that-- in vitro, you have an issue. 904 00:42:25,350 --> 00:42:27,630 And there's not much you can do about it, 905 00:42:27,630 --> 00:42:30,540 except control the relative concentration of the metals. 906 00:42:30,540 --> 00:42:32,010 Inside the cell, do you think it's 907 00:42:32,010 --> 00:42:34,410 easy to control the relative concentrations of metals? 908 00:42:34,410 --> 00:42:35,160 What do you think? 909 00:42:37,500 --> 00:42:39,990 Concentration is everything in biology, 910 00:42:39,990 --> 00:42:41,850 we just don't talk about it that much. 911 00:42:41,850 --> 00:42:43,320 Do you think it's easy to-- 912 00:42:43,320 --> 00:42:49,980 say I threw in the outside of a cell 15 millimolar copper. 913 00:42:49,980 --> 00:42:52,147 Do you think the cell could control that? 914 00:42:52,147 --> 00:42:53,730 Do you think it would all get taken in 915 00:42:53,730 --> 00:42:56,640 and then all of your enzymes would be loaded with copper? 916 00:42:59,790 --> 00:43:00,380 No. 917 00:43:00,380 --> 00:43:04,760 So you have to have a way to actually control all of that. 918 00:43:04,760 --> 00:43:07,760 There was a spectacular paper, I think, 919 00:43:07,760 --> 00:43:10,085 published a couple of years ago that sort 920 00:43:10,085 --> 00:43:11,420 of demonstrates this point. 921 00:43:11,420 --> 00:43:14,120 And so I'm going to give you this example, because I 922 00:43:14,120 --> 00:43:19,650 think it really-- it was published in 2009. 923 00:43:19,650 --> 00:43:20,210 So in vivo. 924 00:43:23,030 --> 00:43:24,995 So this would be, over here, in vitro-- 925 00:43:31,800 --> 00:43:32,670 sorry. 926 00:43:32,670 --> 00:43:35,370 In vitro. 927 00:43:35,370 --> 00:43:37,552 And we can't get rid of the in vitro part. 928 00:43:37,552 --> 00:43:39,510 That's the chemical properties of the molecule, 929 00:43:39,510 --> 00:43:40,900 we're stuck with them. 930 00:43:40,900 --> 00:43:51,550 So it depends-- in vivo, metallation 931 00:43:51,550 --> 00:43:53,170 depends on abundance. 932 00:43:57,050 --> 00:43:58,650 Can we control abundance? 933 00:43:58,650 --> 00:44:00,258 Absolutely, we can control abundance. 934 00:44:00,258 --> 00:44:01,800 You've already seen with cholesterol, 935 00:44:01,800 --> 00:44:04,110 you control abundance with transcription factors. 936 00:44:04,110 --> 00:44:05,077 That's one way. 937 00:44:05,077 --> 00:44:05,910 There are many ways. 938 00:44:05,910 --> 00:44:08,400 We're going to see-- that's one of two general ways 939 00:44:08,400 --> 00:44:11,460 that iron is controlled. 940 00:44:11,460 --> 00:44:15,023 What about speciation? 941 00:44:15,023 --> 00:44:17,190 I've already told you-- and we're going to come back 942 00:44:17,190 --> 00:44:19,740 to this with iron later on-- 943 00:44:19,740 --> 00:44:22,440 you know, are the metals all bound to waters? 944 00:44:22,440 --> 00:44:24,270 But you have ATP inside the cell. 945 00:44:24,270 --> 00:44:28,670 Could iron two be bound to ATP? 946 00:44:28,670 --> 00:44:30,410 Absolutely. 947 00:44:30,410 --> 00:44:31,832 So it's a question of competition 948 00:44:31,832 --> 00:44:33,290 and what the binding constants are, 949 00:44:33,290 --> 00:44:36,727 which is what are talking about in recitations this week. 950 00:44:36,727 --> 00:44:38,310 You know, if it's really weakly bound, 951 00:44:38,310 --> 00:44:40,430 then something else will out compete it. 952 00:44:40,430 --> 00:44:42,620 But you can purify-- 953 00:44:42,620 --> 00:44:44,510 you put ATP in a solution, you'll 954 00:44:44,510 --> 00:44:47,720 pick up iron all the time if you do atomic absorption on it. 955 00:44:47,720 --> 00:44:49,490 Because iron can easily bind to all the 956 00:44:49,490 --> 00:44:52,220 negative charges on ATP. 957 00:44:52,220 --> 00:44:56,150 And the other thing that you need to think about 958 00:44:56,150 --> 00:44:57,640 is location. 959 00:44:57,640 --> 00:45:01,050 So location is what we're going to be focused on 960 00:45:01,050 --> 00:45:01,760 in the example. 961 00:45:01,760 --> 00:45:04,850 And what do I mean by location? 962 00:45:04,850 --> 00:45:07,550 Even in a bacteria you have location, right? 963 00:45:07,550 --> 00:45:09,600 What are the two different compartments? 964 00:45:09,600 --> 00:45:13,220 You have a periplasm and you have the cytosol. 965 00:45:13,220 --> 00:45:18,565 We're going to be talking about periplasm, 966 00:45:18,565 --> 00:45:19,440 and you have cytosol. 967 00:45:22,940 --> 00:45:25,880 In us, we have much more complicated locations 968 00:45:25,880 --> 00:45:27,140 in metal homeostasis. 969 00:45:27,140 --> 00:45:29,750 We'll see when we get to the second lecture. 970 00:45:29,750 --> 00:45:32,930 Has a lot of issues it has to deal with, OK? 971 00:45:32,930 --> 00:45:34,280 Let's look at this example. 972 00:45:34,280 --> 00:45:37,520 And I'm not going to spend a lot of time on it, 973 00:45:37,520 --> 00:45:39,770 but let me just show you what you need to think about. 974 00:45:39,770 --> 00:45:47,060 And these workers we're interested in a cyanobacteria. 975 00:45:47,060 --> 00:45:52,040 And they wanted to find what was the protein that bound the most 976 00:45:52,040 --> 00:45:55,970 copper, and what was the protein that bound the most manganese. 977 00:45:55,970 --> 00:46:02,340 So we're looking at two extremes of the Irving-Williams series. 978 00:46:02,340 --> 00:46:05,300 So this group identified-- 979 00:46:08,960 --> 00:46:11,960 C-- I can never remember the acronym. 980 00:46:11,960 --> 00:46:21,920 CucA is the most abundant copper two binder. 981 00:46:21,920 --> 00:46:23,930 And they identified MncA. 982 00:46:27,290 --> 00:46:29,180 And the way they did it was pretty creative. 983 00:46:29,180 --> 00:46:31,410 If you're interested, you can go read the paper. 984 00:46:31,410 --> 00:46:34,940 It's the most abundant manganese binder. 985 00:46:37,910 --> 00:46:39,950 Both of these things are made in the cytosol-- 986 00:46:39,950 --> 00:46:43,088 both of the proteins are made in the cytosol of the cell. 987 00:46:43,088 --> 00:46:44,630 And what they found when they studied 988 00:46:44,630 --> 00:46:50,600 this system in more detail is the structures of the proteins 989 00:46:50,600 --> 00:46:54,290 and the ligands bound to the metals are exactly the same. 990 00:46:54,290 --> 00:46:58,850 So you have a beta barrel in both cases, 991 00:46:58,850 --> 00:47:08,450 and you have the same first coordination sphere. 992 00:47:08,450 --> 00:47:12,140 The first coordination sphere of the ligands 993 00:47:12,140 --> 00:47:13,430 directly bound to the metal. 994 00:47:21,870 --> 00:47:28,590 If you took these two proteins, and you wanted to load MncA-- 995 00:47:28,590 --> 00:47:30,780 this is manganese binder-- 996 00:47:30,780 --> 00:47:34,830 in the test tube, you would have to add 997 00:47:34,830 --> 00:47:39,270 10,000 times more manganese than copper 998 00:47:39,270 --> 00:47:41,730 to get the manganese in there. 999 00:47:41,730 --> 00:47:43,350 So that, again, goes back to this-- 1000 00:47:43,350 --> 00:47:45,558 I mean, it's going to be different for every system-- 1001 00:47:45,558 --> 00:47:47,250 but it goes back to this question 1002 00:47:47,250 --> 00:47:50,670 of controlling metallation inside the cell, which 1003 00:47:50,670 --> 00:47:52,720 is extremely challenging to do. 1004 00:47:52,720 --> 00:47:55,380 And we don't-- there's a major, in my opinion, 1005 00:47:55,380 --> 00:47:58,980 on cell problem in biology. 1006 00:47:58,980 --> 00:48:01,030 They're going to do this by localization. 1007 00:48:01,030 --> 00:48:02,880 So let me just walk you through the kinds 1008 00:48:02,880 --> 00:48:05,370 of experiments they did. 1009 00:48:05,370 --> 00:48:09,930 So, both these two proteins-- the one that binds copper 1010 00:48:09,930 --> 00:48:12,123 and the one that binds manganese-- 1011 00:48:12,123 --> 00:48:13,290 are produced in the cytosol. 1012 00:48:16,530 --> 00:48:19,740 It turns out that the one that binds manganese 1013 00:48:19,740 --> 00:48:24,060 folds uniquely in the cytosol. 1014 00:48:24,060 --> 00:48:27,000 And the cytosol, if you look at metal speciation, 1015 00:48:27,000 --> 00:48:28,830 how much free-- 1016 00:48:28,830 --> 00:48:30,705 you're going to learn about free today, 1017 00:48:30,705 --> 00:48:34,170 or tomorrow in recitation-- how much free copper or zinc do you 1018 00:48:34,170 --> 00:48:35,322 have-- 1019 00:48:35,322 --> 00:48:36,780 do you think you have in the cells? 1020 00:48:36,780 --> 00:48:38,943 Copper two and zinc two in the cell? 1021 00:48:38,943 --> 00:48:40,110 Do you think you have a lot? 1022 00:48:40,110 --> 00:48:41,737 A little. 1023 00:48:41,737 --> 00:48:44,070 AUDIENCE: For copper, I know it's less than one percent. 1024 00:48:44,070 --> 00:48:45,653 JOANNE STUBBE: Yeah, it's less-- yeah. 1025 00:48:45,653 --> 00:48:46,380 It's tiny. 1026 00:48:46,380 --> 00:48:50,400 Both copper and zinc bind extremely tightly to-- 1027 00:48:50,400 --> 00:48:51,930 again, it's all about speciation, 1028 00:48:51,930 --> 00:48:56,210 so it depends on what the ligands are inside the cell. 1029 00:48:56,210 --> 00:48:59,590 And in fact, in the cytosol cyanobacteria, 1030 00:48:59,590 --> 00:49:03,750 they have measured a micromolar of free manganese. 1031 00:49:03,750 --> 00:49:06,870 And so again, this speaks to this question of is manganese 1032 00:49:06,870 --> 00:49:07,800 readily oxidized? 1033 00:49:07,800 --> 00:49:08,520 No. 1034 00:49:08,520 --> 00:49:11,670 So you don't have to worry about reactive oxygen species 1035 00:49:11,670 --> 00:49:13,440 with manganese. 1036 00:49:13,440 --> 00:49:17,115 What happens is this protein folds 1037 00:49:17,115 --> 00:49:18,240 in the cytosol of the cell. 1038 00:49:18,240 --> 00:49:19,350 Comes off the ribosome. 1039 00:49:19,350 --> 00:49:21,720 It picks up the manganese and folds. 1040 00:49:21,720 --> 00:49:25,800 But its location is in the periplasm. 1041 00:49:25,800 --> 00:49:27,600 How does it get to the periplasm? 1042 00:49:27,600 --> 00:49:29,940 It gets-- there are two ways you can get proteins 1043 00:49:29,940 --> 00:49:32,730 from the cytosol to the periplasm. 1044 00:49:32,730 --> 00:49:35,430 One is through the Tat transporter. 1045 00:49:35,430 --> 00:49:38,250 And Tat transfers-- it recognizes 1046 00:49:38,250 --> 00:49:40,320 a couple of arginines. 1047 00:49:40,320 --> 00:49:43,690 A little zip code-- we've seen zip codes over and over again-- 1048 00:49:43,690 --> 00:49:45,630 which then takes it in the folded state 1049 00:49:45,630 --> 00:49:47,257 into the periplasm. 1050 00:49:47,257 --> 00:49:49,590 And the manganese, once it's in there, doesn't come out. 1051 00:49:49,590 --> 00:49:50,400 Doesn't exchange. 1052 00:49:50,400 --> 00:49:52,680 So there's something about the environment 1053 00:49:52,680 --> 00:49:54,790 that does not allow exchange. 1054 00:49:54,790 --> 00:49:59,940 So the manganese is placed into the protein in the cytosol. 1055 00:49:59,940 --> 00:50:03,780 Now, what happens to the copper binding protein? 1056 00:50:03,780 --> 00:50:08,730 In this case, as soon as it comes off the ribosome, 1057 00:50:08,730 --> 00:50:12,180 it gets grabbed by a second kind of transporter. 1058 00:50:12,180 --> 00:50:14,000 And this second kind of transporter 1059 00:50:14,000 --> 00:50:21,070 transfers the unfolded protein through the plasma membrane. 1060 00:50:21,070 --> 00:50:24,400 And it folds in the periplasm. 1061 00:50:24,400 --> 00:50:25,950 And in the periplasm, I don't know 1062 00:50:25,950 --> 00:50:27,720 what the ratio of copper to manganese 1063 00:50:27,720 --> 00:50:30,990 is, but remember-- copper, by this model, 1064 00:50:30,990 --> 00:50:33,330 out competes manganese by a lot. 1065 00:50:33,330 --> 00:50:35,810 So even if you have manganese and copper in equal amounts, 1066 00:50:35,810 --> 00:50:38,460 the copper will always win out. 1067 00:50:38,460 --> 00:50:42,750 And so what happens here is the copper then binds. 1068 00:50:42,750 --> 00:50:46,950 And so the copper is loaded in a different location 1069 00:50:46,950 --> 00:50:48,130 than the manganese. 1070 00:50:48,130 --> 00:50:51,510 So the way this organism-- this is just one solution-- 1071 00:50:51,510 --> 00:50:53,520 I think a pretty creative solution-- 1072 00:50:53,520 --> 00:50:57,930 to how you deal with the Irving-Williams series, which 1073 00:50:57,930 --> 00:51:00,220 we're faced with all the time with 1074 00:51:00,220 --> 00:51:02,700 the many, many metallocofactors we actually 1075 00:51:02,700 --> 00:51:05,850 have inside the cell. 1076 00:51:05,850 --> 00:51:08,190 I'm going to come back-- next time I'll 1077 00:51:08,190 --> 00:51:10,980 talk about two more issues. 1078 00:51:10,980 --> 00:51:13,620 I want you to be in tune with me when 1079 00:51:13,620 --> 00:51:16,320 we move on in the iron world. 1080 00:51:16,320 --> 00:51:20,790 And talk about sort of a big cartoon for metal homeostasis. 1081 00:51:20,790 --> 00:51:23,640 Doesn't matter what the metal is-- any of these metals. 1082 00:51:23,640 --> 00:51:27,080 And then we're going to move on and focus on iron.