1 00:00:00,500 --> 00:00:02,290 The following content is provided 2 00:00:02,290 --> 00:00:06,360 by MIT OpenCourseWare under a Creative Commons license. 3 00:00:06,360 --> 00:00:08,390 Additional information about our license 4 00:00:08,390 --> 00:00:10,800 and MIT OpenCourseWare in general 5 00:00:10,800 --> 00:00:12,196 is available at ocw.mit.edu. 6 00:00:17,749 --> 00:00:19,290 PROFESSOR: Today, we're going to talk 7 00:00:19,290 --> 00:00:25,080 about a very exciting subject regarding molecules. 8 00:00:25,080 --> 00:00:31,770 And this subject has to do with metals in biology. 9 00:00:31,770 --> 00:00:34,330 Metals in biology, by the way, is actually 10 00:00:34,330 --> 00:00:36,490 the name of one of the Gordon research conferences. 11 00:00:36,490 --> 00:00:38,740 So if you get excited about this topic, 12 00:00:38,740 --> 00:00:42,450 you can go to the Gordon research conference web page 13 00:00:42,450 --> 00:00:45,360 and apply to attend the conference next summer 14 00:00:45,360 --> 00:00:48,070 and learn about the latest developments in metals 15 00:00:48,070 --> 00:00:48,570 in biology. 16 00:00:54,480 --> 00:00:56,640 One of the things I want to impart to you today 17 00:00:56,640 --> 00:00:59,130 has to do with where we usually find 18 00:00:59,130 --> 00:01:02,840 metals inside of biomolecules. 19 00:01:02,840 --> 00:01:05,519 So that leads me to talk a little bit 20 00:01:05,519 --> 00:01:06,540 about nature's ligands. 21 00:01:22,640 --> 00:01:27,650 In biological chemistry, we find that molecules sometimes 22 00:01:27,650 --> 00:01:29,100 are quite a bit larger than we're 23 00:01:29,100 --> 00:01:33,910 used to looking at in synthetic small-molecule chemistry. 24 00:01:33,910 --> 00:01:36,720 So I'm going to talk about nature's ligands today 25 00:01:36,720 --> 00:01:41,620 at sort of two levels of size. 26 00:01:41,620 --> 00:01:43,720 Going to talk first about the porphyrin ligand. 27 00:01:51,990 --> 00:01:55,365 And then next, we're going to talk about proteins. 28 00:02:01,260 --> 00:02:02,110 OK. 29 00:02:02,110 --> 00:02:03,990 First this porphyrin ligand is one 30 00:02:03,990 --> 00:02:08,669 of the most pervasive small-molecule hosts for metal 31 00:02:08,669 --> 00:02:11,260 ions within biomolecules. 32 00:02:11,260 --> 00:02:15,010 And it's really quite a remarkable ligand type. 33 00:02:15,010 --> 00:02:18,220 And I'd like you to appreciate it 34 00:02:18,220 --> 00:02:22,020 at the level of being able to draw the structure of it 35 00:02:22,020 --> 00:02:27,780 and to see just how this system can 36 00:02:27,780 --> 00:02:30,680 provide a host for metal atoms. 37 00:02:30,680 --> 00:02:35,310 It is a system that has four nitrogens that it 38 00:02:35,310 --> 00:02:36,870 can donate to a metal center. 39 00:02:36,870 --> 00:02:39,390 So it's a tetradentate ligand. 40 00:02:39,390 --> 00:02:42,150 We talked about bidentate ligands earlier 41 00:02:42,150 --> 00:02:43,300 in the semester. 42 00:02:43,300 --> 00:02:46,110 This is a tetradentate ligand that nature 43 00:02:46,110 --> 00:02:50,220 uses to hold metal ions very tightly where it wants them 44 00:02:50,220 --> 00:02:51,880 in a biomolecule. 45 00:02:51,880 --> 00:02:54,470 And first you draw the four nitrogens 46 00:02:54,470 --> 00:02:56,430 at the corners of a square. 47 00:02:56,430 --> 00:03:02,250 And now we're drawing in the carbon parts of the molecule. 48 00:03:02,250 --> 00:03:08,970 You can draw four vertical lines like that and four 49 00:03:08,970 --> 00:03:12,150 horizontal lines like this. 50 00:03:12,150 --> 00:03:16,180 And then what we're going to do is, at each of the four corners 51 00:03:16,180 --> 00:03:19,100 of this square-shaped molecule, we're 52 00:03:19,100 --> 00:03:22,340 going to go ahead and complete five-membered rings. 53 00:03:29,130 --> 00:03:30,330 Like that. 54 00:03:30,330 --> 00:03:32,700 And then finally, we have four more carbons 55 00:03:32,700 --> 00:03:34,510 to add to this structure. 56 00:03:34,510 --> 00:03:40,930 And those four carbons are at the top, bottom, and left, 57 00:03:40,930 --> 00:03:44,310 and right, like this. 58 00:03:44,310 --> 00:03:44,810 OK. 59 00:03:44,810 --> 00:03:47,430 So you can see that the porphyrin ligand is 60 00:03:47,430 --> 00:03:51,740 a large ring-shaped macrocycle. 61 00:03:51,740 --> 00:03:53,470 And where can a metal ion go? 62 00:03:53,470 --> 00:03:57,120 It can go right in the center. 63 00:03:57,120 --> 00:04:00,060 If you are going to figure oxidation states that involved 64 00:04:00,060 --> 00:04:01,560 a porphyrin molecule, then you would 65 00:04:01,560 --> 00:04:04,550 need to know the charge like we know that one chloride is 66 00:04:04,550 --> 00:04:05,520 in a metal complex. 67 00:04:05,520 --> 00:04:06,850 It carries a one minus charge. 68 00:04:06,850 --> 00:04:09,660 And you need to know how many charges the porphyrin 69 00:04:09,660 --> 00:04:11,897 ligand would carry if a metal were in there, 70 00:04:11,897 --> 00:04:13,480 so that you could figure the oxidation 71 00:04:13,480 --> 00:04:15,771 state and the d-electron count for the metal that would 72 00:04:15,771 --> 00:04:17,870 be inserted into the porphyrin. 73 00:04:17,870 --> 00:04:21,089 And to do that, we first recognize 74 00:04:21,089 --> 00:04:26,580 that if you isolate a porphyrin without the metal in there, 75 00:04:26,580 --> 00:04:31,150 then two of the four nitrogens have hydrogens attached. 76 00:04:31,150 --> 00:04:31,680 OK? 77 00:04:31,680 --> 00:04:36,960 And also this is a system-- you'll remember the graphite 78 00:04:36,960 --> 00:04:39,340 structure that we looked at last time that 79 00:04:39,340 --> 00:04:42,170 had all these p orbitals perpendicular to the plane 80 00:04:42,170 --> 00:04:43,360 of the molecule. 81 00:04:43,360 --> 00:04:46,030 And similarly, this is an unsaturated molecule 82 00:04:46,030 --> 00:04:50,470 that has lots of pi bonding going on with the carbon 83 00:04:50,470 --> 00:04:52,640 p orbitals that are perpendicular to the plane 84 00:04:52,640 --> 00:04:54,080 of the blackboard here. 85 00:04:54,080 --> 00:04:56,830 So it can be a quite flat molecule, but as you'll see 86 00:04:56,830 --> 00:04:58,980 it can ruffle as well when it carries out 87 00:04:58,980 --> 00:05:00,910 its biological function. 88 00:05:00,910 --> 00:05:04,560 And so we'll go ahead and draw in the double bonds. 89 00:05:04,560 --> 00:05:08,850 And this is the part where people sometimes get messed up 90 00:05:08,850 --> 00:05:10,540 because one thing we don't want to do 91 00:05:10,540 --> 00:05:14,220 is draw five bonds to a carbon atom. 92 00:05:14,220 --> 00:05:14,970 OK? 93 00:05:14,970 --> 00:05:17,011 And if you see a structure that's drawn that way, 94 00:05:17,011 --> 00:05:19,300 you know that someone has made a mistake because they 95 00:05:19,300 --> 00:05:21,270 violated the octet rule. 96 00:05:21,270 --> 00:05:21,910 All right? 97 00:05:21,910 --> 00:05:25,140 So let's draw in, starting here at the upper left, 98 00:05:25,140 --> 00:05:31,610 we'll put in a double bond here, and here, and here. 99 00:05:31,610 --> 00:05:34,620 And then this is where it gets interesting 100 00:05:34,620 --> 00:05:38,390 because I could put a double bond here now or here 101 00:05:38,390 --> 00:05:40,830 because this carbon's going to need a double bond. 102 00:05:40,830 --> 00:05:43,220 I'm going to choose to put it here, 103 00:05:43,220 --> 00:05:46,630 and then I'm going to use symmetry across the way 104 00:05:46,630 --> 00:05:49,200 to put one there. 105 00:05:49,200 --> 00:05:57,916 And having done that, I can now go here, here, here, here, 106 00:05:57,916 --> 00:06:06,740 and-- so I didn't use symmetry across the way. 107 00:06:06,740 --> 00:06:10,760 This is much easier to do and it's small. 108 00:06:10,760 --> 00:06:11,990 OK. 109 00:06:11,990 --> 00:06:15,230 So now you can see that these two nitrogens here 110 00:06:15,230 --> 00:06:18,380 would have lone pairs that point into the center 111 00:06:18,380 --> 00:06:23,570 of the porphyrin ligand and same across the way 112 00:06:23,570 --> 00:06:26,090 by symmetry there. 113 00:06:26,090 --> 00:06:33,100 And then when we remove these two hydrogens as H plus 114 00:06:33,100 --> 00:06:37,120 and insert a metal ion, you'll see that the porphyrin is 115 00:06:37,120 --> 00:06:40,450 actually behaving as a dianion. 116 00:06:44,500 --> 00:06:49,310 When a metal ion and-- most typically, 117 00:06:49,310 --> 00:06:52,930 we're going to see that the metal that 118 00:06:52,930 --> 00:06:55,650 appears at the center of a porphyrin ligand 119 00:06:55,650 --> 00:06:57,600 is an iron center. 120 00:06:57,600 --> 00:07:01,892 And so the nitrogens provide four lone pairs 121 00:07:01,892 --> 00:07:04,350 in the plane of the molecule that are directed at the metal 122 00:07:04,350 --> 00:07:04,850 center. 123 00:07:04,850 --> 00:07:08,380 So that's four out of the six positions for an octahedron 124 00:07:08,380 --> 00:07:09,950 all in the same plane. 125 00:07:09,950 --> 00:07:13,190 And because of the rigid delocalized structure 126 00:07:13,190 --> 00:07:16,810 of the porphyrin ligand, what we find 127 00:07:16,810 --> 00:07:19,850 is that a metal center that is in a porphyrin 128 00:07:19,850 --> 00:07:22,030 is very difficult to take out of the porphyrin 129 00:07:22,030 --> 00:07:26,540 because these lone pairs are forced at all times 130 00:07:26,540 --> 00:07:30,050 to converge on the same point which is the metal ion. 131 00:07:30,050 --> 00:07:32,200 OK. 132 00:07:32,200 --> 00:07:35,550 You know that I enjoy giving lectures using shock. 133 00:07:35,550 --> 00:07:39,390 But sometimes when the molecules are really big, 134 00:07:39,390 --> 00:07:45,040 it's easier to use a computer to portray them quickly. 135 00:07:45,040 --> 00:07:48,520 And that's what we're going to do today, 136 00:07:48,520 --> 00:07:51,700 assuming my computer participates and cooperates 137 00:07:51,700 --> 00:07:52,410 with me today. 138 00:07:56,310 --> 00:07:59,950 And what I really want to do is to go through and show 139 00:07:59,950 --> 00:08:07,450 you a tour of six really important metalloproteins. 140 00:08:07,450 --> 00:08:09,400 And all of these, you'll see the heme 141 00:08:09,400 --> 00:08:14,050 plays an important role, the iron in a porphyrin. 142 00:08:14,050 --> 00:08:19,710 This unit together, when an iron is located inside 143 00:08:19,710 --> 00:08:21,705 of a porphyrin, that is referred to as heme. 144 00:08:26,820 --> 00:08:28,460 So you can have other metals in there, 145 00:08:28,460 --> 00:08:30,126 and then it's not called a heme anymore, 146 00:08:30,126 --> 00:08:33,409 but that would be very similar structurally. 147 00:08:33,409 --> 00:08:35,090 OK. 148 00:08:35,090 --> 00:08:40,260 So I'm going to take advantage of some really nice summaries 149 00:08:40,260 --> 00:08:43,960 that have been put together on the protein data bank website. 150 00:08:43,960 --> 00:08:46,610 So since the first crystal structure 151 00:08:46,610 --> 00:08:53,550 was determined for a protein, the structural information 152 00:08:53,550 --> 00:08:56,180 has been collected and put together in one site 153 00:08:56,180 --> 00:08:58,020 so that researchers all over the world 154 00:08:58,020 --> 00:09:00,140 can take advantage of this information 155 00:09:00,140 --> 00:09:03,620 to advance science and medicine. 156 00:09:03,620 --> 00:09:09,300 And since 2000, the protein data bank 157 00:09:09,300 --> 00:09:12,690 has been collecting information about certain biomolecules 158 00:09:12,690 --> 00:09:17,960 and putting out one new little short story or vignette 159 00:09:17,960 --> 00:09:18,740 each month. 160 00:09:18,740 --> 00:09:21,430 So you have their Molecule of the Month 161 00:09:21,430 --> 00:09:23,265 portion of the protein data bank website. 162 00:09:26,244 --> 00:09:27,660 So each month, you can go and look 163 00:09:27,660 --> 00:09:30,240 and see which new molecule has been added 164 00:09:30,240 --> 00:09:33,270 and learn more about it. 165 00:09:33,270 --> 00:09:37,630 And I want to start out here with myoglobin. 166 00:09:42,520 --> 00:09:43,300 OK. 167 00:09:43,300 --> 00:09:49,800 And I'm going to give you this website information online 168 00:09:49,800 --> 00:09:51,990 associated with today's notes. 169 00:09:51,990 --> 00:09:55,240 So you're going to see that myoglobin was the first protein 170 00:09:55,240 --> 00:09:56,730 structure. 171 00:09:56,730 --> 00:10:00,940 There are many different ways to represent protein molecules. 172 00:10:00,940 --> 00:10:04,780 Protein chains are composed of linear polimers 173 00:10:04,780 --> 00:10:08,292 of the naturally occurring amino acids with polypeptide chains. 174 00:10:08,292 --> 00:10:10,500 And they can sometimes be very, very large in length, 175 00:10:10,500 --> 00:10:13,600 and then they fold up and make three-dimensional shapes. 176 00:10:13,600 --> 00:10:16,690 And these are big ligands for metal ions. 177 00:10:16,690 --> 00:10:18,890 And what you're going to see is that in many 178 00:10:18,890 --> 00:10:24,170 of the most important proteins, the place where the reaction's 179 00:10:24,170 --> 00:10:26,440 actually happened is that the metal center that's 180 00:10:26,440 --> 00:10:29,330 embedded somewhere in the protein 181 00:10:29,330 --> 00:10:33,510 chain as it has folded up around it to modulate function. 182 00:10:33,510 --> 00:10:34,010 OK. 183 00:10:34,010 --> 00:10:37,400 So this is a pretty small protein-- myoglobin is. 184 00:10:37,400 --> 00:10:40,870 It's an oxygen storage protein. 185 00:10:40,870 --> 00:10:45,100 So as you know, diatomic molecules like dioxygen 186 00:10:45,100 --> 00:10:49,460 are important for life on Earth, and O2 in particular-- 187 00:10:49,460 --> 00:10:52,760 not only do we have hemoglobin that we'll talk about next, 188 00:10:52,760 --> 00:10:57,310 but there are also oxygen storage molecules 189 00:10:57,310 --> 00:10:59,470 like this myoglobin molecule. 190 00:10:59,470 --> 00:11:03,710 And this was the first structure that was determined, 191 00:11:03,710 --> 00:11:05,820 and that happened in 1960. 192 00:11:05,820 --> 00:11:07,900 So we've been getting information 193 00:11:07,900 --> 00:11:11,540 at the molecular level about how biomolecules work really 194 00:11:11,540 --> 00:11:14,960 since 1960 using x-ray diffraction techniques. 195 00:11:14,960 --> 00:11:18,160 So first, a worker in this area has 196 00:11:18,160 --> 00:11:21,730 to obtain a large quantity of the protein in question, 197 00:11:21,730 --> 00:11:25,630 get it purified, and then find conditions under which it will 198 00:11:25,630 --> 00:11:28,830 grow single crystals so that you can impinge 199 00:11:28,830 --> 00:11:31,000 upon it with an x-ray beam and then looked 200 00:11:31,000 --> 00:11:35,020 at the intensities of the diffracted x-ray beams 201 00:11:35,020 --> 00:11:37,730 and then work back to solve the crystal structure 202 00:11:37,730 --> 00:11:40,980 and find out what arrangement of atoms in 3D space 203 00:11:40,980 --> 00:11:44,100 could have given rise to that pattern of diffraction 204 00:11:44,100 --> 00:11:45,070 intensities. 205 00:11:45,070 --> 00:11:47,840 And that was first done for a protein in 1960. 206 00:11:47,840 --> 00:11:50,660 You might find it interesting that this protein 207 00:11:50,660 --> 00:11:53,511 was isolated from sperm whales. 208 00:11:53,511 --> 00:11:54,010 OK? 209 00:11:54,010 --> 00:11:55,570 Sperm whale muscles. 210 00:11:55,570 --> 00:11:58,816 And in order to get a protein pure, as I said, 211 00:11:58,816 --> 00:11:59,690 you need a lot of it. 212 00:11:59,690 --> 00:12:03,730 And the muscles of sperm whales are very large indeed, 213 00:12:03,730 --> 00:12:06,550 and so these workers were able to collect 214 00:12:06,550 --> 00:12:11,140 large amounts of this red protein myoglobin 215 00:12:11,140 --> 00:12:12,980 from sperm whale muscles in order 216 00:12:12,980 --> 00:12:14,830 to finally get enough in pure form 217 00:12:14,830 --> 00:12:17,190 to crystallize and solve the crystal structure. 218 00:12:17,190 --> 00:12:22,640 And since this is involved in oxygen storage 219 00:12:22,640 --> 00:12:26,570 and you know that whales and other marine mammals 220 00:12:26,570 --> 00:12:29,900 can dive below the ocean surface for long periods of time, 221 00:12:29,900 --> 00:12:31,970 they need to store lots of oxygen 222 00:12:31,970 --> 00:12:34,680 so that they can use their muscles during the length 223 00:12:34,680 --> 00:12:35,650 of a long dive. 224 00:12:35,650 --> 00:12:38,940 And this protein is very abundant in muscles. 225 00:12:38,940 --> 00:12:42,457 It gives meat its red color and so forth. 226 00:12:42,457 --> 00:12:44,290 The color is coming from the heme units that 227 00:12:44,290 --> 00:12:46,290 are embedded in the myoglobin. 228 00:12:46,290 --> 00:12:49,460 And I'll step through a little bit of this, 229 00:12:49,460 --> 00:12:52,250 but I would like to actually show 230 00:12:52,250 --> 00:12:55,050 you some more of the features of this. 231 00:12:55,050 --> 00:12:56,900 You'll see as you go through this website, 232 00:12:56,900 --> 00:13:00,530 there are both static and interactive ways 233 00:13:00,530 --> 00:13:02,700 to look at metalloproteins. 234 00:13:02,700 --> 00:13:05,420 And sometimes the polypeptide chain 235 00:13:05,420 --> 00:13:07,340 composed of the amino acids is just 236 00:13:07,340 --> 00:13:09,400 represented here as sort of a tube, 237 00:13:09,400 --> 00:13:11,390 so you can see the polymer has wrapped up. 238 00:13:11,390 --> 00:13:13,600 And here they've chosen to represent 239 00:13:13,600 --> 00:13:17,340 the heme unit as spheres to highlight it and accentuate it. 240 00:13:17,340 --> 00:13:21,270 And in this first structure of myoglobin, 241 00:13:21,270 --> 00:13:24,680 there was not an O2 molecule bound to the heme unit. 242 00:13:24,680 --> 00:13:26,660 Instead, there was a water molecule 243 00:13:26,660 --> 00:13:29,800 bound to the iron center of the heme unit. 244 00:13:29,800 --> 00:13:34,120 And then down here, they rotate the structure perpendicular 245 00:13:34,120 --> 00:13:38,750 so you can appreciate the square nature of the heme unit 246 00:13:38,750 --> 00:13:43,440 together with when viewed edge on, its flat nature. 247 00:13:43,440 --> 00:13:44,785 So that's the structure here. 248 00:13:44,785 --> 00:13:52,380 And you can see how this myoglobin molecule, 249 00:13:52,380 --> 00:13:54,760 this big protein polymer, has folded up 250 00:13:54,760 --> 00:13:59,470 like a clam to grasp this heme unit that inside of it 251 00:13:59,470 --> 00:14:06,550 will bind O2 and store it when oxygen comes to it. 252 00:14:06,550 --> 00:14:09,990 And so later structures of myoglobin 253 00:14:09,990 --> 00:14:13,640 have been carried out that do show you the position of the O2 254 00:14:13,640 --> 00:14:15,000 molecule. 255 00:14:15,000 --> 00:14:20,200 And in these two representations that we are given here, 256 00:14:20,200 --> 00:14:22,820 we first see, once again, the heme unit 257 00:14:22,820 --> 00:14:26,680 as spheres and the protein side chain just as tubes. 258 00:14:26,680 --> 00:14:29,080 And then this representation in the bottom, 259 00:14:29,080 --> 00:14:32,430 all the protein side chain atoms themselves 260 00:14:32,430 --> 00:14:33,590 are represented as spheres. 261 00:14:33,590 --> 00:14:36,130 And you can see that when that's the case, 262 00:14:36,130 --> 00:14:38,960 you can tell that the atoms of the protein 263 00:14:38,960 --> 00:14:42,040 ligand really fill up space very well. 264 00:14:42,040 --> 00:14:44,340 And what they're showing you with this circle here 265 00:14:44,340 --> 00:14:48,720 is the location beneath that side chain of the dioxygen 266 00:14:48,720 --> 00:14:49,765 molecule inside there. 267 00:14:49,765 --> 00:14:51,140 And so they're trying to give you 268 00:14:51,140 --> 00:14:56,700 the idea that when O2 is on the heme unit inside the myoglobin, 269 00:14:56,700 --> 00:15:00,440 the myoglobin protein matrix actually covers that up. 270 00:15:00,440 --> 00:15:04,960 And so in order for the O2 molecule to get into the heme 271 00:15:04,960 --> 00:15:09,270 and also to exit from the metalloprotein, 272 00:15:09,270 --> 00:15:10,850 this thing has to be flexible enough 273 00:15:10,850 --> 00:15:13,900 to open up and allow the dioxygen molecule 274 00:15:13,900 --> 00:15:16,960 to exit from its chamber inside the metalloprotein. 275 00:15:20,840 --> 00:15:22,620 OK. 276 00:15:22,620 --> 00:15:25,370 Now next, I would like to go over to the site 277 00:15:25,370 --> 00:15:28,675 where we're actually able to look at things interactively. 278 00:15:37,980 --> 00:15:41,240 And you need to be able to enter the code 279 00:15:41,240 --> 00:15:42,415 for the protein in question. 280 00:15:46,500 --> 00:15:51,750 And so this is a structure of the myoglobin molecule 281 00:15:51,750 --> 00:15:55,840 that was obtained at molecular or atomic resolution. 282 00:15:55,840 --> 00:15:57,650 So one of the parameters that you'll 283 00:15:57,650 --> 00:16:00,560 see when people discuss protein structures 284 00:16:00,560 --> 00:16:04,670 is the resolution to which the data have permitted 285 00:16:04,670 --> 00:16:06,240 the structure to be determined. 286 00:16:06,240 --> 00:16:09,310 And the best quality structures, the ones 287 00:16:09,310 --> 00:16:11,725 where we know most precisely where the atoms are, 288 00:16:11,725 --> 00:16:16,440 are the ones that have very low resolution numbers. 289 00:16:16,440 --> 00:16:22,240 In other words, this one, if you looked at the introductory web 290 00:16:22,240 --> 00:16:24,470 page there so that the resolution was 291 00:16:24,470 --> 00:16:26,800 at the level of one angstrom. 292 00:16:26,800 --> 00:16:27,300 OK? 293 00:16:27,300 --> 00:16:32,250 So we're talking on the order of carbon-carbon bond distances 294 00:16:32,250 --> 00:16:34,920 so that you can actually see all the individual atoms quite 295 00:16:34,920 --> 00:16:35,860 clearly. 296 00:16:35,860 --> 00:16:38,360 And this has to do with the quality of the crystal 297 00:16:38,360 --> 00:16:44,660 and how large was the angle at which you could collect spots 298 00:16:44,660 --> 00:16:48,390 from the diffracted x-rays as they came off of the crystal. 299 00:16:48,390 --> 00:16:50,450 And so this is nice. 300 00:16:50,450 --> 00:16:52,780 This one requires Java. 301 00:16:52,780 --> 00:16:55,062 That's why I was not able to use Athena today. 302 00:16:55,062 --> 00:16:56,520 But actually, I come in here, and I 303 00:16:56,520 --> 00:16:58,400 see that the Athena terminal is down anyway, 304 00:16:58,400 --> 00:17:01,810 so this was a good choice as it turns out. 305 00:17:01,810 --> 00:17:05,010 And this is nice because you can actually 306 00:17:05,010 --> 00:17:06,295 zoom on these structures. 307 00:17:08,900 --> 00:17:12,050 One thing you notice is that this was crystallized 308 00:17:12,050 --> 00:17:14,339 with sulfate present in the medium, 309 00:17:14,339 --> 00:17:15,900 so there was some buffer present. 310 00:17:15,900 --> 00:17:19,359 And it had sulfate, and the sulfates crystallized together 311 00:17:19,359 --> 00:17:20,410 with the protein chains. 312 00:17:20,410 --> 00:17:23,260 And as we look at representations of proteins, 313 00:17:23,260 --> 00:17:25,619 you'll appreciate that this representation 314 00:17:25,619 --> 00:17:32,040 shows the polypeptide backbone as these ribbons. 315 00:17:32,040 --> 00:17:36,050 And that emphasizes that when proteins fold and go 316 00:17:36,050 --> 00:17:38,280 into a three-dimensional shape that they 317 00:17:38,280 --> 00:17:41,530 need for their function, they can usually 318 00:17:41,530 --> 00:17:44,640 do so either by making coils like this 319 00:17:44,640 --> 00:17:48,650 that look like springs-- these are called helices. 320 00:17:48,650 --> 00:17:52,640 Or they can just form random coils as you can see out here. 321 00:17:52,640 --> 00:17:56,140 And oftentimes you have these helices 322 00:17:56,140 --> 00:17:58,990 that are like long springy tubes that are then 323 00:17:58,990 --> 00:18:03,470 connected at their termini by coils of the protein side 324 00:18:03,470 --> 00:18:04,160 chain. 325 00:18:04,160 --> 00:18:09,760 And then, of course, here embedded within this structure 326 00:18:09,760 --> 00:18:11,690 is our heme unit. 327 00:18:11,690 --> 00:18:15,410 And one thing if you look at this one pretty closely, 328 00:18:15,410 --> 00:18:18,990 you'll be able to see that this heme is a little more 329 00:18:18,990 --> 00:18:20,760 interesting, a little more complicated, 330 00:18:20,760 --> 00:18:23,240 than the one that I drew on the board 331 00:18:23,240 --> 00:18:25,700 here because the one I drew on the board 332 00:18:25,700 --> 00:18:28,260 was assumed to have just hydrogens at each 333 00:18:28,260 --> 00:18:31,840 of these carbon positions at the periphery of the molecule. 334 00:18:31,840 --> 00:18:34,030 But when we look at the one that's actually 335 00:18:34,030 --> 00:18:38,420 been found in myoglobin at atomic resolutions-- 336 00:18:38,420 --> 00:18:43,210 so you see this one is actually with the O2 molecule present. 337 00:18:43,210 --> 00:18:48,670 For some reason, no bond drawn there, but that's OK. 338 00:18:48,670 --> 00:18:51,000 We've talked a lot about molecular representations, 339 00:18:51,000 --> 00:18:52,624 and we understand what's going on here. 340 00:18:52,624 --> 00:18:54,900 It's that people have decided that we would represent 341 00:18:54,900 --> 00:18:57,741 the atoms over here without any detail at all just 342 00:18:57,741 --> 00:18:58,740 showing them as ribbons. 343 00:18:58,740 --> 00:19:01,073 And then here we're going to look at the atom positions. 344 00:19:01,073 --> 00:19:02,860 And you can see the five-membered ring 345 00:19:02,860 --> 00:19:06,740 here, here, here, and here at each nitrogen 346 00:19:06,740 --> 00:19:08,670 corner of the heme unit. 347 00:19:08,670 --> 00:19:10,870 There's the iron center. 348 00:19:10,870 --> 00:19:14,260 And then, notice that the substituents, 349 00:19:14,260 --> 00:19:16,280 instead of being just hydrogen or organic, 350 00:19:16,280 --> 00:19:17,540 there's a carbon here. 351 00:19:17,540 --> 00:19:19,700 This looks like a methyl group perhaps. 352 00:19:19,700 --> 00:19:23,560 And this one looks like an ethyl group or maybe a vinyl group 353 00:19:23,560 --> 00:19:27,050 here if this is one of the etioporphyrin ligand types. 354 00:19:27,050 --> 00:19:30,770 And then coming out here, we have CH2, CH2. 355 00:19:30,770 --> 00:19:33,610 We have something that looks like it might 356 00:19:33,610 --> 00:19:35,700 be a ketone residue since we have 357 00:19:35,700 --> 00:19:37,470 a single oxygen on a carbon there, 358 00:19:37,470 --> 00:19:42,640 or alternatively, it could be some other oxygenated residue. 359 00:19:42,640 --> 00:19:45,440 But over here, you see that there's 360 00:19:45,440 --> 00:19:47,620 a link to this five-membered ring of the heme. 361 00:19:47,620 --> 00:19:51,540 You have a couple of methylenes and then a carboxylate residue. 362 00:19:51,540 --> 00:19:55,175 And so when you investigate these structures, 363 00:19:55,175 --> 00:20:00,410 these metal complexes that are embedded in the protein 364 00:20:00,410 --> 00:20:03,320 usually are held in position, both by the way 365 00:20:03,320 --> 00:20:06,090 the protein folds up around the heme unit, 366 00:20:06,090 --> 00:20:10,230 but also because, in this case, the heme unit is connected 367 00:20:10,230 --> 00:20:13,360 to residues like this carboxylic acid residue that 368 00:20:13,360 --> 00:20:18,040 can hydrogen bond to specific residues on the protein side 369 00:20:18,040 --> 00:20:18,540 chain. 370 00:20:18,540 --> 00:20:21,940 So if we were to look at the atoms involved in the side 371 00:20:21,940 --> 00:20:24,980 chain right over here that's next to this carboxylate 372 00:20:24,980 --> 00:20:29,490 residue, this probably has some hydrogen bond acceptor 373 00:20:29,490 --> 00:20:32,570 that is interacting with this carboxylate residue-- and same 374 00:20:32,570 --> 00:20:36,480 over here-- to hold this heme unit in its desired position 375 00:20:36,480 --> 00:20:40,080 or in its required position for function. 376 00:20:40,080 --> 00:20:42,780 So please keep that in mind as you go ahead 377 00:20:42,780 --> 00:20:46,720 and look at different structures in the database. 378 00:20:46,720 --> 00:21:00,560 And now let's go to the 2003 description of hemoglobin. 379 00:21:00,560 --> 00:21:01,435 This is rather nice. 380 00:21:06,330 --> 00:21:06,830 OK. 381 00:21:06,830 --> 00:21:10,725 So myoglobin was a pretty small protein having a single heme 382 00:21:10,725 --> 00:21:12,700 unit. 383 00:21:12,700 --> 00:21:16,620 And hemoglobin is much more interesting. 384 00:21:16,620 --> 00:21:20,170 It actually has four big pieces that come together, 385 00:21:20,170 --> 00:21:22,280 and it has four heme units in it. 386 00:21:27,431 --> 00:21:27,930 OK. 387 00:21:27,930 --> 00:21:29,720 And as you go through this website, 388 00:21:29,720 --> 00:21:33,390 you'll find out about why blood is red versus blue. 389 00:21:33,390 --> 00:21:38,590 It's actually red in the oxygenated form and blue 390 00:21:38,590 --> 00:21:41,090 in the deoxygenated form. 391 00:21:41,090 --> 00:21:43,110 And there are many crystal structures 392 00:21:43,110 --> 00:21:45,650 of different hemoglobins and mutants of hemoglobins 393 00:21:45,650 --> 00:21:49,880 in the database and both with and without the oxygen 394 00:21:49,880 --> 00:21:50,920 molecules. 395 00:21:50,920 --> 00:21:53,840 There's a discussion here of artificial blood. 396 00:21:53,840 --> 00:21:56,160 And as you'll see what the premise is 397 00:21:56,160 --> 00:21:58,920 here for the design of artificial blood 398 00:21:58,920 --> 00:22:00,920 motivated by a desire to have blood 399 00:22:00,920 --> 00:22:05,030 available for transfusions and so forth that would not 400 00:22:05,030 --> 00:22:06,920 be contaminated, would be just synthetic-- 401 00:22:06,920 --> 00:22:09,770 would be just pure hemoglobin in fact. 402 00:22:09,770 --> 00:22:14,380 But what happens is that without the protective casing 403 00:22:14,380 --> 00:22:16,890 of the red blood cell, the four parts of the hemoglobin 404 00:22:16,890 --> 00:22:18,830 molecule fall apart from each other, 405 00:22:18,830 --> 00:22:21,060 and they don't do their function properly anymore. 406 00:22:21,060 --> 00:22:23,226 And we'll learn more about the function in a moment. 407 00:22:23,226 --> 00:22:26,110 So people have actually made mutants 408 00:22:26,110 --> 00:22:33,160 wherein you make covalent bonds between the four 409 00:22:33,160 --> 00:22:34,675 different subunits of the hemoglobin 410 00:22:34,675 --> 00:22:36,830 so that they can't fall apart. 411 00:22:36,830 --> 00:22:39,390 And that's one of the approaches to the synthesis 412 00:22:39,390 --> 00:22:42,790 of artificial blood. 413 00:22:42,790 --> 00:22:47,690 And then this is a rather nice little video 414 00:22:47,690 --> 00:22:53,420 which comes from two different hemoglobin crystal structures. 415 00:22:53,420 --> 00:22:56,762 If you imagine different crystal structures, 416 00:22:56,762 --> 00:22:58,470 like crystal structure of hemoglobin when 417 00:22:58,470 --> 00:23:02,360 it has O2 bound verses when it does not have O2 bound, 418 00:23:02,360 --> 00:23:05,070 as snapshots of the molecule in motion, 419 00:23:05,070 --> 00:23:06,570 then you can generate a little movie 420 00:23:06,570 --> 00:23:09,600 like this one shown here from the experimental data 421 00:23:09,600 --> 00:23:11,150 on the crystal structure. 422 00:23:11,150 --> 00:23:14,380 And so the really remarkable thing here 423 00:23:14,380 --> 00:23:17,220 is that-- see when the O2 molecule is bound 424 00:23:17,220 --> 00:23:21,060 to the heme, then the protein itself, 425 00:23:21,060 --> 00:23:26,710 this large unit that is comprised of four parts, as one 426 00:23:26,710 --> 00:23:29,560 confirmation, one structure, and it changes quite a bit. 427 00:23:29,560 --> 00:23:32,700 You see how much it is moving around when the dioxygen 428 00:23:32,700 --> 00:23:34,840 molecule comes off of the heme. 429 00:23:34,840 --> 00:23:38,190 And that is related very much to its function. 430 00:23:38,190 --> 00:23:42,650 In fact, it turns out that, in hemoglobin, 431 00:23:42,650 --> 00:23:47,860 when the deoxyhemoglobin arrives at a place in your body 432 00:23:47,860 --> 00:23:53,400 where there is a lot of oxygen, the first O2 molecule binding 433 00:23:53,400 --> 00:23:57,050 event is very difficult to achieve because it involves 434 00:23:57,050 --> 00:24:00,890 a big change in the structure of the entire protein molecule. 435 00:24:00,890 --> 00:24:03,600 But once that first O2 molecule has bound, 436 00:24:03,600 --> 00:24:07,050 the whole protein molecule has changed its structure such 437 00:24:07,050 --> 00:24:10,260 that the next three hemes take up their O2 molecules 438 00:24:10,260 --> 00:24:13,400 rapidly in rapid fire succession after the first one has 439 00:24:13,400 --> 00:24:14,040 done it. 440 00:24:14,040 --> 00:24:17,420 So the heme goes into a place of high-oxygen concentration 441 00:24:17,420 --> 00:24:19,660 and it rapidly loads up with four O2's. 442 00:24:19,660 --> 00:24:21,140 And then it moves on. 443 00:24:21,140 --> 00:24:25,830 And then when it gets to a location that needs oxygen, 444 00:24:25,830 --> 00:24:28,130 the first one comes off, and then the next three fire 445 00:24:28,130 --> 00:24:32,031 off in rapid succession because of the change in the protein 446 00:24:32,031 --> 00:24:32,530 shape. 447 00:24:32,530 --> 00:24:34,760 And so hemoglobin is, therefore, very 448 00:24:34,760 --> 00:24:38,730 good at collecting four O2 molecules at a time 449 00:24:38,730 --> 00:24:42,280 and then delivering them to where they're needed. 450 00:24:42,280 --> 00:24:48,760 And this is just a beautiful example of the way protein 451 00:24:48,760 --> 00:24:51,490 crystallography can teach us about mechanisms 452 00:24:51,490 --> 00:24:53,540 of biomolecules. 453 00:24:53,540 --> 00:24:54,180 OK. 454 00:24:54,180 --> 00:25:03,390 And then also you can learn about disease mechanisms 455 00:25:03,390 --> 00:25:05,960 in the data bank here because this 456 00:25:05,960 --> 00:25:10,010 is from a different crystal structure where 457 00:25:10,010 --> 00:25:12,660 a mutation has occurred in the protein side 458 00:25:12,660 --> 00:25:13,670 chain of the hemoglobin. 459 00:25:13,670 --> 00:25:16,030 So this is one hemoglobin molecule. 460 00:25:16,030 --> 00:25:19,950 And it turns out that when that mutation is present, 461 00:25:19,950 --> 00:25:22,500 unfortunately, it causes hemoglobin molecules 462 00:25:22,500 --> 00:25:25,730 to aggregate and stick together and clump together like that. 463 00:25:25,730 --> 00:25:30,400 And that phenomenon is associated with diseases 464 00:25:30,400 --> 00:25:32,185 like sickle cell anemia. 465 00:25:32,185 --> 00:25:32,685 OK? 466 00:25:32,685 --> 00:25:34,810 So the structure of the protein is 467 00:25:34,810 --> 00:25:38,950 changed when one residue of the amino acid side chain 468 00:25:38,950 --> 00:25:41,100 is changed to a different amino acid. 469 00:25:41,100 --> 00:25:43,510 And then these things all aggregate together 470 00:25:43,510 --> 00:25:48,500 instead of forming nice disks as normal red blood cells do. 471 00:25:48,500 --> 00:25:51,890 They stick together and form these different shapes 472 00:25:51,890 --> 00:25:57,240 that are not good at functioning the way they're supposed to. 473 00:25:57,240 --> 00:26:02,590 And then here is a close-up on where the action takes place 474 00:26:02,590 --> 00:26:04,650 in the hemoglobin molecule. 475 00:26:04,650 --> 00:26:07,420 And this, again, is from the two crystal structures 476 00:26:07,420 --> 00:26:09,330 that we were talking about-- the one that 477 00:26:09,330 --> 00:26:12,650 has the oxygen molecule bound to the iron of the hemoglobin 478 00:26:12,650 --> 00:26:14,220 and the one that does not. 479 00:26:14,220 --> 00:26:16,860 And one thing that you'll notice is 480 00:26:16,860 --> 00:26:21,430 that when the O2 molecules is absent, this iron in yellow 481 00:26:21,430 --> 00:26:24,480 represented here as this yellow sphere is sort of dipping 482 00:26:24,480 --> 00:26:27,440 down on one side of the hemoglobin-- 483 00:26:27,440 --> 00:26:31,580 the heme plane toward the nitrogen of this histidine. 484 00:26:31,580 --> 00:26:35,790 So this is part of the amino acid protein side chain. 485 00:26:35,790 --> 00:26:40,160 And a histidine residue has this Lewis space group here that 486 00:26:40,160 --> 00:26:43,340 is coordinated to the iron. 487 00:26:43,340 --> 00:26:48,230 And so in that form, this is a five-coordinate metal center 488 00:26:48,230 --> 00:26:51,550 with five ligands of square pyramidal geometry. 489 00:26:51,550 --> 00:26:55,300 It's an octahedron missing one of those six ligands. 490 00:26:55,300 --> 00:26:58,980 And then when the O2 ligand comes in and binds and becomes 491 00:26:58,980 --> 00:27:00,540 the sixth ligand in the coordination 492 00:27:00,540 --> 00:27:03,220 sphere of the iron, the iron responds to that 493 00:27:03,220 --> 00:27:07,490 by moving up to the other side of the porphyrin plane, 494 00:27:07,490 --> 00:27:10,630 and it draws with it the histidine ligand. 495 00:27:10,630 --> 00:27:13,910 So the iron moving up pulls the histidine up. 496 00:27:13,910 --> 00:27:17,620 And that structural change at the iron center 497 00:27:17,620 --> 00:27:20,460 is then propagated throughout the amino acid side 498 00:27:20,460 --> 00:27:22,510 chain to which this is connected and then, 499 00:27:22,510 --> 00:27:24,370 ultimately, throughout the entire protein 500 00:27:24,370 --> 00:27:27,380 to give you that huge conformational change that we 501 00:27:27,380 --> 00:27:30,470 saw when O2 is bound versus when it's not. 502 00:27:30,470 --> 00:27:32,870 So the coordination chemistry of the iron 503 00:27:32,870 --> 00:27:38,590 here is really controlling the peptide confirmation 504 00:27:38,590 --> 00:27:42,230 and the function of this protein. 505 00:27:42,230 --> 00:27:42,730 OK. 506 00:27:42,730 --> 00:27:46,440 So these bio molecules actually accumulate 507 00:27:46,440 --> 00:27:48,660 quite a lot of the concepts that we've 508 00:27:48,660 --> 00:27:49,980 talked about all semester. 509 00:27:49,980 --> 00:27:53,170 And that phenomenon is going to continue 510 00:27:53,170 --> 00:27:55,310 as I take you through a tour of a couple more 511 00:27:55,310 --> 00:27:57,160 of these important molecules. 512 00:27:57,160 --> 00:27:58,620 Now the first one was a small one 513 00:27:58,620 --> 00:28:00,590 that we looked at, myoglobin, and I'm 514 00:28:00,590 --> 00:28:02,680 going to continue that sort of progression 515 00:28:02,680 --> 00:28:06,890 by now talking about another small protein. 516 00:28:06,890 --> 00:28:10,975 This one may even remind you in its appearance of myoglobin. 517 00:28:10,975 --> 00:28:16,010 But instead of serving to store O2, 518 00:28:16,010 --> 00:28:18,750 like myoglobin, cytochrome c, which 519 00:28:18,750 --> 00:28:22,020 has a single heme unit in it here, 520 00:28:22,020 --> 00:28:25,930 serves as a shuttle for electrons. 521 00:28:25,930 --> 00:28:26,530 OK? 522 00:28:26,530 --> 00:28:29,870 The pathway that electrons take in the whole process 523 00:28:29,870 --> 00:28:35,980 of respiration in organisms is really quite fascinating. 524 00:28:35,980 --> 00:28:38,130 We require reducing agents. 525 00:28:38,130 --> 00:28:39,340 We eat food. 526 00:28:39,340 --> 00:28:40,780 Those are reducing agents. 527 00:28:40,780 --> 00:28:44,840 And then we go ahead and burn that fuel 528 00:28:44,840 --> 00:28:47,510 using the oxygen molecule. 529 00:28:47,510 --> 00:28:49,040 And that's where we get our energy. 530 00:28:49,040 --> 00:28:56,460 We don't burn the fuel literally by having us burst into flames, 531 00:28:56,460 --> 00:28:59,025 but we do it in a controlled manner, in a stepwise manner. 532 00:28:59,025 --> 00:29:01,250 And we take advantage of those steps 533 00:29:01,250 --> 00:29:03,830 to carry out the processes of life. 534 00:29:03,830 --> 00:29:06,650 And so there are proteins like these small cytochrome 535 00:29:06,650 --> 00:29:10,270 c proteins that are developed expressly 536 00:29:10,270 --> 00:29:12,440 for the purpose of moving electrons from one 537 00:29:12,440 --> 00:29:15,020 place in the body to another. 538 00:29:15,020 --> 00:29:18,540 So it's like a little electron shuttle protein. 539 00:29:18,540 --> 00:29:22,870 And these are just two views of it. 540 00:29:25,450 --> 00:29:28,370 And if you read this part of the website, 541 00:29:28,370 --> 00:29:31,910 you'll also learn that this one has remained 542 00:29:31,910 --> 00:29:33,760 virtually unchanged over eons. 543 00:29:33,760 --> 00:29:37,240 It's a very ancient protein. 544 00:29:37,240 --> 00:29:39,945 OK. 545 00:29:39,945 --> 00:29:45,740 And this picture shows you cytochrome c protein 546 00:29:45,740 --> 00:29:48,910 and where it picks up its electrons 547 00:29:48,910 --> 00:29:51,320 and then where it goes to drop them off. 548 00:29:51,320 --> 00:29:55,400 So here's an enormous protein relative 549 00:29:55,400 --> 00:29:58,050 to cytochrome-- cytochrome c is the little teeny one here 550 00:29:58,050 --> 00:29:59,940 with just a single heme unit. 551 00:29:59,940 --> 00:30:01,120 OK? 552 00:30:01,120 --> 00:30:02,070 Look at this thing. 553 00:30:02,070 --> 00:30:06,270 This thing is-- I don't know what this looks like, 554 00:30:06,270 --> 00:30:10,810 but it looks like a bizarre-shaped entity, 555 00:30:10,810 --> 00:30:11,420 shall we say. 556 00:30:11,420 --> 00:30:16,490 And this yellow stripe that goes across the page 557 00:30:16,490 --> 00:30:19,800 here is actually a membrane. 558 00:30:19,800 --> 00:30:24,140 So proteins are often classified as to whether they 559 00:30:24,140 --> 00:30:27,190 are membrane proteins-- meaning that they don't move around. 560 00:30:27,190 --> 00:30:29,730 But they're embedded or fixed in a membrane 561 00:30:29,730 --> 00:30:33,344 with one part of their molecule on one side of the membrane 562 00:30:33,344 --> 00:30:34,760 and the other part of the molecule 563 00:30:34,760 --> 00:30:36,259 up on the other side of the membrane 564 00:30:36,259 --> 00:30:38,070 and then the part of the molecule 565 00:30:38,070 --> 00:30:41,700 that's actually located within the membrane, and then 566 00:30:41,700 --> 00:30:43,800 non-membrane proteins like cytochrome c 567 00:30:43,800 --> 00:30:47,360 that are actually mobile and can move about within a cell. 568 00:30:47,360 --> 00:30:50,960 And cytochrome c goes over to this large protein 569 00:30:50,960 --> 00:30:53,510 which has lots of different cofactors shown here 570 00:30:53,510 --> 00:30:58,310 in red which incidentally are hemes themselves. 571 00:30:58,310 --> 00:31:02,030 And so this molecule-- the big protein 572 00:31:02,030 --> 00:31:05,680 here embedded in the matrix is called cytochrome bc1. 573 00:31:05,680 --> 00:31:08,920 And it is a protein involved in this process of respiration. 574 00:31:08,920 --> 00:31:10,640 It is producing electrons. 575 00:31:10,640 --> 00:31:13,780 And cytochrome c comes over here, and as you can see, 576 00:31:13,780 --> 00:31:16,370 it fits really well into this spot right here 577 00:31:16,370 --> 00:31:18,250 on cytochrome bc1. 578 00:31:18,250 --> 00:31:21,800 And that's a feature of protein-protein interactions 579 00:31:21,800 --> 00:31:25,490 that they often contain residues on their surface 580 00:31:25,490 --> 00:31:28,265 that are hydrogen bonding residues, for example, 581 00:31:28,265 --> 00:31:29,890 that make their surfaces complimentary. 582 00:31:29,890 --> 00:31:32,770 And it means that when cytochrome c comes in 583 00:31:32,770 --> 00:31:38,170 into cytochrome bc1, it actually locks in in one particular way. 584 00:31:38,170 --> 00:31:42,790 And this complex between cytochrome c and cytochrome bc1 585 00:31:42,790 --> 00:31:43,867 has been crystallized. 586 00:31:43,867 --> 00:31:45,950 And the crystal structure of it's in the database, 587 00:31:45,950 --> 00:31:48,716 and that's where we get this representation shown here. 588 00:31:48,716 --> 00:31:51,340 So you can actually go through-- if you're a crystallographer-- 589 00:31:51,340 --> 00:31:55,130 and look at all the different complimentary interactions that 590 00:31:55,130 --> 00:31:58,740 holds cytochrome c into place on cytochrome bc1 591 00:31:58,740 --> 00:32:00,380 when it docks there. 592 00:32:00,380 --> 00:32:02,130 And what I'm going to tell you in a moment 593 00:32:02,130 --> 00:32:04,840 is that as electrons are originating 594 00:32:04,840 --> 00:32:08,130 within cytochrome bc1 here, they have 595 00:32:08,130 --> 00:32:10,240 a pathway through which they move 596 00:32:10,240 --> 00:32:12,000 and they get up into this heme unit. 597 00:32:12,000 --> 00:32:15,840 And then when cytochrome c docs, the heme unit within it 598 00:32:15,840 --> 00:32:18,270 can get close enough to this heme unit 599 00:32:18,270 --> 00:32:20,460 that their wave functions can overlap, 600 00:32:20,460 --> 00:32:22,280 and the electron can tunnel right 601 00:32:22,280 --> 00:32:25,310 across into the cytochrome c which accepts it. 602 00:32:25,310 --> 00:32:28,140 And then it goes off, and it's looking for this protein 603 00:32:28,140 --> 00:32:30,860 next which has also been crystallized and characterized 604 00:32:30,860 --> 00:32:31,632 in the database. 605 00:32:31,632 --> 00:32:32,840 And we'll talk about it next. 606 00:32:32,840 --> 00:32:35,950 This is called cytochrome C oxidase 607 00:32:35,950 --> 00:32:40,030 because it oxidizes the reduced form of cytochrome c. 608 00:32:40,030 --> 00:32:44,949 And let me just say that probably one 609 00:32:44,949 --> 00:32:46,490 thing we won't have on the final will 610 00:32:46,490 --> 00:32:51,840 be working the molecular orbital diagram of this molecule. 611 00:32:51,840 --> 00:32:54,270 As you can see, we've got up to a position 612 00:32:54,270 --> 00:32:55,670 here where the number of orbitals 613 00:32:55,670 --> 00:32:57,770 is kind of prohibitive to do that sort of thing. 614 00:32:57,770 --> 00:33:00,880 Now you can understand why the computational chemists have 615 00:33:00,880 --> 00:33:02,370 quite a big challenge as they try 616 00:33:02,370 --> 00:33:05,790 to understand at a molecular level all of the processes 617 00:33:05,790 --> 00:33:10,120 of life and, really, a long way from doing that actually. 618 00:33:10,120 --> 00:33:12,070 When people attempt to do that these days, 619 00:33:12,070 --> 00:33:16,850 they're usually making some big approximations with the protein 620 00:33:16,850 --> 00:33:20,540 side chain because that's the electronically uninteresting 621 00:33:20,540 --> 00:33:21,900 part of the system. 622 00:33:21,900 --> 00:33:25,240 Where the metals are is where everything is happening. 623 00:33:25,240 --> 00:33:31,520 Let's go down here and see what else we can learn. 624 00:33:31,520 --> 00:33:32,370 OK. 625 00:33:32,370 --> 00:33:34,970 Here's a close-up with a different representation 626 00:33:34,970 --> 00:33:37,730 of when cytochrome c molecule docs on 627 00:33:37,730 --> 00:33:40,880 to the cytochrome bc1 complex. 628 00:33:40,880 --> 00:33:44,860 And here we've got the polypeptide chain of cytochrome 629 00:33:44,860 --> 00:33:49,820 c represented as these pink tubes, and then 630 00:33:49,820 --> 00:33:53,640 as these yellow tubes down here, this little part 631 00:33:53,640 --> 00:33:56,170 of the cytochrome bc1 molecule that is reaching up 632 00:33:56,170 --> 00:33:58,730 to interact with cytochrome c. 633 00:33:58,730 --> 00:34:03,310 And here's the heme unit of cytochrome bc1 634 00:34:03,310 --> 00:34:05,780 that is right at the surface of that protein 635 00:34:05,780 --> 00:34:08,909 so that it can overlap it's wave function with the heme unit 636 00:34:08,909 --> 00:34:14,080 of cytochrome c to permit an electron to transfer and reduce 637 00:34:14,080 --> 00:34:15,030 cytochrome c. 638 00:34:18,222 --> 00:34:19,139 OK. 639 00:34:19,139 --> 00:34:29,550 And so now we'll go on to cytochrome c oxidase. 640 00:34:44,820 --> 00:34:45,320 OK. 641 00:34:45,320 --> 00:34:49,679 So in reading about cytochrome c oxidase, 642 00:34:49,679 --> 00:34:51,420 you're going to learn more about oxygen. 643 00:34:51,420 --> 00:34:55,446 And this piece of today's story, together 644 00:34:55,446 --> 00:34:56,820 with the one that I'm going to go 645 00:34:56,820 --> 00:34:59,650 to next, which will be with respect to photosystem 646 00:34:59,650 --> 00:35:03,200 one and photosystem two, are sort of the two ends 647 00:35:03,200 --> 00:35:05,090 of the chain of respiration. 648 00:35:05,090 --> 00:35:09,590 Because with photosynthesis, that we'll talk about next, 649 00:35:09,590 --> 00:35:13,580 we use light energy to create O2. 650 00:35:13,580 --> 00:35:18,120 And most organisms on the planet have these cytochrome so 651 00:35:18,120 --> 00:35:20,020 that they can use O2. 652 00:35:20,020 --> 00:35:21,750 In fact, what they do is they take 653 00:35:21,750 --> 00:35:24,630 electrons that are derived from food 654 00:35:24,630 --> 00:35:29,800 and get energy by combining them with oxygen, 655 00:35:29,800 --> 00:35:33,560 reducing them-- reducing the oxygen molecule-- to water, 656 00:35:33,560 --> 00:35:35,760 and at the same time pushing protons 657 00:35:35,760 --> 00:35:38,110 across a membrane which stores energy 658 00:35:38,110 --> 00:35:42,240 that can be used for other processes like building up ATP. 659 00:35:42,240 --> 00:35:46,030 And so, the cytochrome c oxidase is getting its electrons 660 00:35:46,030 --> 00:35:49,510 from cytochrome c and it's using those electrons 661 00:35:49,510 --> 00:35:52,330 to reduce the O2 molecule. 662 00:35:52,330 --> 00:35:56,170 And for that reason, cytochrome c oxidase 663 00:35:56,170 --> 00:35:59,510 should have somewhere in it a place for the dioxygen 664 00:35:59,510 --> 00:36:01,030 molecule to bind. 665 00:36:01,030 --> 00:36:04,190 Just like is the case for hemoglobin. 666 00:36:04,190 --> 00:36:07,820 We found out where oxygen binds in hemoglobin. 667 00:36:07,820 --> 00:36:09,640 And notice that as you go through they'll 668 00:36:09,640 --> 00:36:14,020 make an analogy here to charging of a battery 669 00:36:14,020 --> 00:36:17,740 or actually of a capacitor as you're reducing the O2 670 00:36:17,740 --> 00:36:20,720 molecules with these electrons coming in from the cytochrome c 671 00:36:20,720 --> 00:36:24,350 shuttle, and making water and pushing protons 672 00:36:24,350 --> 00:36:28,110 across a membrane that is being likened to charging a battery 673 00:36:28,110 --> 00:36:30,660 or charging a capacitor. 674 00:36:30,660 --> 00:36:32,320 And this is a membrane-bound protein 675 00:36:32,320 --> 00:36:34,530 just like the bc1 complexes. 676 00:36:34,530 --> 00:36:37,690 And here's a nice graphic that shows a number 677 00:36:37,690 --> 00:36:39,360 of the important cofactors. 678 00:36:39,360 --> 00:36:41,410 A cofactor is just something that's 679 00:36:41,410 --> 00:36:44,970 an integral part of a protein but isn't 680 00:36:44,970 --> 00:36:48,980 part of the amino acid backbone of the polypeptide chain. 681 00:36:48,980 --> 00:36:50,800 And usually it's something like a heme, 682 00:36:50,800 --> 00:36:55,300 but here not only do we have hemes, but here's a heme that 683 00:36:55,300 --> 00:36:57,810 is thought to be involved in the electron transport 684 00:36:57,810 --> 00:37:00,930 chain within the cytochrome C oxidase molecule. 685 00:37:00,930 --> 00:37:04,270 And over here is a heme that is thought 686 00:37:04,270 --> 00:37:07,970 to be important in binding the O2 molecule. 687 00:37:07,970 --> 00:37:12,460 And also this one has copper. 688 00:37:12,460 --> 00:37:16,140 So these 3D transition elements are turning out 689 00:37:16,140 --> 00:37:17,770 to be pretty important in biology. 690 00:37:17,770 --> 00:37:20,990 The iron is frequently the site for the binding 691 00:37:20,990 --> 00:37:24,500 of a diatomic molecule, but look at this. 692 00:37:24,500 --> 00:37:27,660 This structure that's in the Cambridge database here, 693 00:37:27,660 --> 00:37:32,400 its PDB entry 1OCO, actually has a carbon monoxide 694 00:37:32,400 --> 00:37:35,520 ligand bonded to the iron in the active site 695 00:37:35,520 --> 00:37:39,240 where O2 is supposed to bind when this enzyme functions. 696 00:37:39,240 --> 00:37:41,460 So this is a poisoned form of the enzyme. 697 00:37:41,460 --> 00:37:44,540 CO comes in and binds and shuts this enzyme down, 698 00:37:44,540 --> 00:37:47,580 and that's what they were able to purify and crystallize 699 00:37:47,580 --> 00:37:49,320 and get the 3D structure of. 700 00:37:49,320 --> 00:37:52,660 And then here you have a heme iron 701 00:37:52,660 --> 00:37:56,010 which interacts with the carbon end of carbon monoxide. 702 00:37:56,010 --> 00:38:00,700 And then positioned over here, and also ligated by residues 703 00:38:00,700 --> 00:38:03,050 from the protein side chain, is copper, 704 00:38:03,050 --> 00:38:04,530 referred to as copper b. 705 00:38:04,530 --> 00:38:05,900 So this is a copper ion. 706 00:38:05,900 --> 00:38:08,140 And that means that when a diatomic molecule 707 00:38:08,140 --> 00:38:10,120 goes in and binds to the heme at one end, 708 00:38:10,120 --> 00:38:13,000 it's other end is binding simultaneously to copper b. 709 00:38:13,000 --> 00:38:14,780 And that diatomic molecule is serving 710 00:38:14,780 --> 00:38:17,850 as a bridge between two metal ions-- the iron 711 00:38:17,850 --> 00:38:20,150 heme and the copper b up here. 712 00:38:20,150 --> 00:38:23,870 And then also up here is a copper 713 00:38:23,870 --> 00:38:25,760 a site, where you have a pair of coppers. 714 00:38:25,760 --> 00:38:29,710 And this site is referred to and thought 715 00:38:29,710 --> 00:38:32,310 to be the port of entry of the O2 molecule 716 00:38:32,310 --> 00:38:34,740 into cytochrome c oxidase. 717 00:38:34,740 --> 00:38:37,730 That O2 somehow comes in and may bind here first, but then 718 00:38:37,730 --> 00:38:39,340 go over here and electrons are coming 719 00:38:39,340 --> 00:38:42,790 in through this other heme unit into the one that binds the O2 720 00:38:42,790 --> 00:38:46,960 and ends up reducing it to generate water. 721 00:38:46,960 --> 00:38:50,810 So that is a really pretty picture 722 00:38:50,810 --> 00:38:53,200 of how complicated the machinery of an enzyme 723 00:38:53,200 --> 00:38:55,970 can be to take advantage of all this lovely transition element 724 00:38:55,970 --> 00:39:02,260 chemistry and redox chemistry to carry out life function. 725 00:39:02,260 --> 00:39:20,910 And also-- let's see-- OK, so what they're doing here 726 00:39:20,910 --> 00:39:22,390 is analyzing this. 727 00:39:22,390 --> 00:39:24,340 This cytochrome c oxidase is composed 728 00:39:24,340 --> 00:39:26,440 of a number of different protein chains 729 00:39:26,440 --> 00:39:28,710 that are all packed together into the overall complex, 730 00:39:28,710 --> 00:39:30,876 and they're coloring each of these residues a little 731 00:39:30,876 --> 00:39:32,250 differently here. 732 00:39:32,250 --> 00:39:35,620 And then when you get down to the bottom of this page, 733 00:39:35,620 --> 00:39:41,100 they're comparing a subunit of cytochrome c oxidase 734 00:39:41,100 --> 00:39:44,480 to an actual cytochrome c oxidase that 735 00:39:44,480 --> 00:39:48,430 is found in bacteria molecules. 736 00:39:48,430 --> 00:39:49,849 Bacteria organisms. 737 00:39:49,849 --> 00:39:51,390 So bacteria have their own cytochrome 738 00:39:51,390 --> 00:39:56,510 c oxidases and this looks like the central core 739 00:39:56,510 --> 00:39:59,220 of our cytochrome c oxidases. 740 00:39:59,220 --> 00:40:03,660 And the idea is that mitochondria in cells 741 00:40:03,660 --> 00:40:07,350 may, at some point way, way back in evolution, 742 00:40:07,350 --> 00:40:10,950 have been the result of bacterial invaders into cells. 743 00:40:10,950 --> 00:40:15,460 And then these bacteria became integral parts of ourselves, 744 00:40:15,460 --> 00:40:18,140 and we added new proteins on and elaborated 745 00:40:18,140 --> 00:40:21,100 to change the function as we deemed necessary 746 00:40:21,100 --> 00:40:21,910 through evolution. 747 00:40:21,910 --> 00:40:26,320 But these ways of comparing structures 748 00:40:26,320 --> 00:40:31,310 of simpler or ancient proteins to ones that are more modern 749 00:40:31,310 --> 00:40:33,770 is an interesting way to learn about how life 750 00:40:33,770 --> 00:40:37,920 has evolved on this planet. 751 00:40:37,920 --> 00:40:41,810 OK, and-- so cytochrome c oxidase 752 00:40:41,810 --> 00:40:43,295 is a pretty remarkable protein. 753 00:40:43,295 --> 00:40:48,070 And I want to see if I can get this thing up interactively 754 00:40:48,070 --> 00:40:51,500 because the structure is quite impressive. 755 00:40:51,500 --> 00:40:56,360 And I'm using this carbon monoxide substituted variant. 756 00:41:06,180 --> 00:41:09,740 When these structures have many thousands of atoms, 757 00:41:09,740 --> 00:41:13,390 sometimes these things take a little while to load. 758 00:41:13,390 --> 00:41:15,108 So hopefully this won't take too long. 759 00:41:21,590 --> 00:41:25,410 The viewer that-- there are actually several viewers 760 00:41:25,410 --> 00:41:28,540 that you can choose from on this protein data bank web page, 761 00:41:28,540 --> 00:41:30,470 and they possess features that allow 762 00:41:30,470 --> 00:41:32,624 you to turn on or turn off different parts 763 00:41:32,624 --> 00:41:33,290 of the molecule. 764 00:41:33,290 --> 00:41:35,450 You can subtract away the cofactors, 765 00:41:35,450 --> 00:41:39,012 or subtract away some of the loops or the helices, 766 00:41:39,012 --> 00:41:41,220 and that allows you to explore the structure in quite 767 00:41:41,220 --> 00:41:42,310 some detail. 768 00:41:42,310 --> 00:41:45,970 Now, each of these are different protein residues, 769 00:41:45,970 --> 00:41:49,870 different residues of this enormous protein. 770 00:41:49,870 --> 00:41:58,980 So let's come down here, zoom in a little bit. 771 00:42:01,980 --> 00:42:15,450 OK, if I notice that-- there I can subtract away 772 00:42:15,450 --> 00:42:24,520 the coils that hold together the various loops, OK? 773 00:42:24,520 --> 00:42:28,810 Or you can take away, in fact, all of those things. 774 00:42:28,810 --> 00:42:34,530 And notice the cofactors themselves are these heme units 775 00:42:34,530 --> 00:42:37,150 and they have some interesting and complicated side chains. 776 00:42:41,180 --> 00:42:42,860 But they're the same sort of heme units 777 00:42:42,860 --> 00:42:48,860 in general that we see in myoglobin or in cytochrome c. 778 00:42:48,860 --> 00:42:51,156 OK? 779 00:42:51,156 --> 00:42:53,430 Let's see . 780 00:42:53,430 --> 00:43:11,060 Actually-- there are orientations 781 00:43:11,060 --> 00:43:13,560 of this molecule that you can access 782 00:43:13,560 --> 00:43:17,040 that shows you how this thing fits nicely into a membrane. 783 00:43:17,040 --> 00:43:18,170 This is one of them. 784 00:43:18,170 --> 00:43:23,050 You see all these different helices that 785 00:43:23,050 --> 00:43:25,270 are aligned with one another. 786 00:43:25,270 --> 00:43:28,680 This is the part of the cytochrome c oxidase 787 00:43:28,680 --> 00:43:31,780 that resides in the membrane. 788 00:43:31,780 --> 00:43:35,620 And all of these residues are lipophilic, 789 00:43:35,620 --> 00:43:39,320 so they interact with the non-polar interior 790 00:43:39,320 --> 00:43:41,810 of the membrane very well allowing this protein 791 00:43:41,810 --> 00:43:45,420 to just stay in as part of the membrane. 792 00:43:45,420 --> 00:43:49,080 And then these loops that extend on one side of the membrane 793 00:43:49,080 --> 00:43:51,660 or onto the other contain polar residues 794 00:43:51,660 --> 00:43:53,870 that would prefer to interact with water 795 00:43:53,870 --> 00:43:56,700 or with charged things in solution. 796 00:43:56,700 --> 00:43:59,590 And so that's an important aspect of the way 797 00:43:59,590 --> 00:44:02,900 a membrane protein like this is the structured. 798 00:44:02,900 --> 00:44:05,320 If I could just take away the loops here, 799 00:44:05,320 --> 00:44:08,820 the coil, that becomes quite clear. 800 00:44:12,750 --> 00:44:15,410 So now, with the few minutes we have left, 801 00:44:15,410 --> 00:44:17,840 I'm going to go ahead and talk about photosystem I 802 00:44:17,840 --> 00:44:20,510 and II at the other end of the chain of respiration 803 00:44:20,510 --> 00:44:22,880 where organisms are actually-- you know, 804 00:44:22,880 --> 00:44:25,639 they realize that eventually you would run out 805 00:44:25,639 --> 00:44:27,930 of all the food on the planet and everything would die. 806 00:44:27,930 --> 00:44:31,420 And so they learned to absorb light 807 00:44:31,420 --> 00:44:36,400 and to use light energy to carry out processes of life 808 00:44:36,400 --> 00:44:38,160 instead of just using the energy that you 809 00:44:38,160 --> 00:44:41,970 get when you add electrons to water, to two oxygen 810 00:44:41,970 --> 00:44:44,500 and make water. 811 00:44:44,500 --> 00:44:53,880 And so let's go look first at photosystem I. 812 00:44:53,880 --> 00:44:57,860 Photosystems I and II are different parts 813 00:44:57,860 --> 00:45:02,240 of the photosynthetic pathway and of the electron utilization 814 00:45:02,240 --> 00:45:04,840 chain, electron transfer chain. 815 00:45:04,840 --> 00:45:07,720 And what you see is that photosystem I and II are both 816 00:45:07,720 --> 00:45:09,380 going to be membrane proteins. 817 00:45:09,380 --> 00:45:11,520 These, incidentally, are usually much harder 818 00:45:11,520 --> 00:45:14,229 to obtain in large quantities and much harder to crystallize, 819 00:45:14,229 --> 00:45:16,270 and their structures much harder to characterize. 820 00:45:16,270 --> 00:45:19,110 So there are a lot of important membrane proteins 821 00:45:19,110 --> 00:45:22,360 whose structures are not very well known yet. 822 00:45:22,360 --> 00:45:24,760 In photosystem I it's a trimer. 823 00:45:24,760 --> 00:45:27,610 There's one piece right here, it's shaped like a big disk. 824 00:45:27,610 --> 00:45:30,070 Here's another piece and here's another piece. 825 00:45:30,070 --> 00:45:31,900 And then if you flip it by 90 degrees 826 00:45:31,900 --> 00:45:34,340 to look at it edge-on you can see here 827 00:45:34,340 --> 00:45:36,260 it is sitting in the membrane. 828 00:45:36,260 --> 00:45:39,480 And these have colorful cofactors. 829 00:45:39,480 --> 00:45:41,930 These now have porphyrins, many porphyrins, 830 00:45:41,930 --> 00:45:47,310 that contain instead of iron, contain magnesium. 831 00:45:47,310 --> 00:45:50,180 And these are the chlorophylls that are present in here 832 00:45:50,180 --> 00:45:53,620 as light gathering entities. 833 00:45:53,620 --> 00:46:04,200 And then-- OK. 834 00:46:04,200 --> 00:46:06,070 OK, with this picture I can tell you 835 00:46:06,070 --> 00:46:09,010 quite a bit about photosystem I. Here, you can see, 836 00:46:09,010 --> 00:46:12,280 is the part of the molecule that is embedded in the membrane. 837 00:46:12,280 --> 00:46:19,550 And what we have is the ability to extract electrons 838 00:46:19,550 --> 00:46:23,420 from a molecule that is hard to oxidize. 839 00:46:23,420 --> 00:46:27,080 OK, so in photosystem II you'll see 840 00:46:27,080 --> 00:46:30,100 that the source of electrons is water. 841 00:46:30,100 --> 00:46:33,460 And so photosystem II is capable of pulling electrons out 842 00:46:33,460 --> 00:46:37,070 of water, generating dioxygen, the most important 843 00:46:37,070 --> 00:46:40,510 photosynthetic reaction on our planet. 844 00:46:40,510 --> 00:46:43,870 In the case of photosystem I, the source of the electrons, 845 00:46:43,870 --> 00:46:47,970 instead of being water, is a little protein 846 00:46:47,970 --> 00:46:49,650 called plastocyanin. 847 00:46:49,650 --> 00:46:55,970 So plastocyanin comes up here and it is oxidized. 848 00:46:55,970 --> 00:46:59,560 And it's a little bit like the LED 849 00:46:59,560 --> 00:47:01,850 we talked about last time, because what 850 00:47:01,850 --> 00:47:09,120 happens is there's a pair of heme units here, 851 00:47:09,120 --> 00:47:12,020 they're not hemes of chlorophylls, the special pair, 852 00:47:12,020 --> 00:47:17,630 that when light energy is absorbed and excitation occurs, 853 00:47:17,630 --> 00:47:19,850 it sends an electron to high energy. 854 00:47:19,850 --> 00:47:23,930 And normally, after such an absorption of light occurs, 855 00:47:23,930 --> 00:47:25,930 that electron would just drop back down 856 00:47:25,930 --> 00:47:28,510 and the molecule would go back to its ground state. 857 00:47:28,510 --> 00:47:31,330 But what this molecule has been built to do 858 00:47:31,330 --> 00:47:33,270 is to take that high energy electron 859 00:47:33,270 --> 00:47:37,490 and descend it through a series of redox active molecules. 860 00:47:37,490 --> 00:47:40,370 And it actually goes all the way through here, 861 00:47:40,370 --> 00:47:44,600 and these are little iron four s four cubes. 862 00:47:44,600 --> 00:47:47,920 So these are beautiful little inorganic molecules, Fe4 S4 863 00:47:47,920 --> 00:47:48,600 cubes. 864 00:47:48,600 --> 00:47:50,650 The electron pathway goes through these redox 865 00:47:50,650 --> 00:47:54,220 active units and jumps from one of these Fe S4 cubes 866 00:47:54,220 --> 00:47:56,960 to another, and finally jumps out to a protein that 867 00:47:56,960 --> 00:48:00,700 docks up here to accept it, which is a ferredoxin protein. 868 00:48:00,700 --> 00:48:04,500 So after light comes in and you get a high energy electron, 869 00:48:04,500 --> 00:48:07,790 you get a hole, the hole is filled through oxidation 870 00:48:07,790 --> 00:48:09,860 in the case of photosystem I and plastocyanin 871 00:48:09,860 --> 00:48:14,700 in the case of photosystem II of water, and so the chain goes. 872 00:48:14,700 --> 00:48:16,580 Now, having run out of time here, 873 00:48:16,580 --> 00:48:20,780 I will talk about photosystem II at the beginning of next hour.