1 00:00:00,720 --> 00:00:10,780 [Music] 2 00:00:16,609 --> 00:00:14,000 thanks Yuichiro and I want to extend my 3 00:00:17,900 --> 00:00:16,619 thanks again to Eric in the scientific 4 00:00:21,170 --> 00:00:17,910 organizing committee for putting 5 00:00:23,120 --> 00:00:21,180 together a great series of talks and I 6 00:00:26,390 --> 00:00:23,130 I'm gonna try to hook hook my talk in 7 00:00:30,410 --> 00:00:26,400 here from from a perspective of trying 8 00:00:32,210 --> 00:00:30,420 to understand ways that we can observe 9 00:00:35,990 --> 00:00:32,220 the historical record of catalytic 10 00:00:39,200 --> 00:00:36,000 evolution through time and this is a 11 00:00:41,479 --> 00:00:39,210 very broad question and I'm going to use 12 00:00:46,270 --> 00:00:41,489 a case study of looking at microbial 13 00:00:48,770 --> 00:00:46,280 sulfate reduction as a system to try to 14 00:00:52,810 --> 00:00:48,780 see if we can do this if we can start to 15 00:00:55,099 --> 00:00:52,820 link enzyme sequence evolution to 16 00:00:57,349 --> 00:00:55,109 fractionation of stable isotopes and be 17 00:00:59,209 --> 00:00:57,359 able to monitor the historical record of 18 00:01:01,880 --> 00:00:59,219 that those isotope fractionation through 19 00:01:03,770 --> 00:01:01,890 time but I'm also interested in doing 20 00:01:05,719 --> 00:01:03,780 this from the perspective of 21 00:01:08,480 --> 00:01:05,729 understanding cells today from a 22 00:01:10,790 --> 00:01:08,490 microbial ecology perspective where we 23 00:01:12,289 --> 00:01:10,800 might be able to use stable isotope 24 00:01:14,480 --> 00:01:12,299 fractionation as a way of gaining 25 00:01:16,280 --> 00:01:14,490 insight onto the physiological state of 26 00:01:20,289 --> 00:01:16,290 a cell that we might have in a bottle or 27 00:01:25,100 --> 00:01:20,299 have in a lake or some other environment 28 00:01:27,710 --> 00:01:25,110 so this is a I'm presenting today but a 29 00:01:31,100 --> 00:01:27,720 lot of the work was done by Minh sub-sub 30 00:01:33,890 --> 00:01:31,110 it was a postdoc previously in my 31 00:01:36,789 --> 00:01:33,900 previous affiliation at Cal Tech he's 32 00:01:40,130 --> 00:01:36,799 now at a Seoul National University and 33 00:01:41,929 --> 00:01:40,140 here at Elsi Chris butch and Mackenzie 34 00:01:47,569 --> 00:01:41,939 Smith are also I'm working towards some 35 00:01:50,060 --> 00:01:47,579 of the goals of this project and so yeah 36 00:01:51,469 --> 00:01:50,070 I titled my talk and and I'm really 37 00:01:53,719 --> 00:01:51,479 going to Center the talk about this idea 38 00:01:57,859 --> 00:01:53,729 of using stable isotopes to understand 39 00:02:00,410 --> 00:01:57,869 cellular physiology but I but I want to 40 00:02:03,230 --> 00:02:00,420 make it a little bit more general than 41 00:02:05,209 --> 00:02:03,240 that and hopefully be able to get some 42 00:02:06,920 --> 00:02:05,219 criticism and comments and other ideas 43 00:02:09,259 --> 00:02:06,930 about how we might be able to use this 44 00:02:12,550 --> 00:02:09,269 approach of looking at stable isotope 45 00:02:15,319 --> 00:02:12,560 fractionation coupled with knowledge of 46 00:02:19,009 --> 00:02:15,329 catalytic kinetics to be able to follow 47 00:02:21,890 --> 00:02:19,019 enzyme evolution of time and so broadly 48 00:02:22,610 --> 00:02:21,900 speaking we have two main repositories 49 00:02:27,199 --> 00:02:22,620 of 50 00:02:30,140 --> 00:02:27,209 one isn't stuff that we can go out and 51 00:02:32,570 --> 00:02:30,150 see like these piles of cells that we 52 00:02:35,990 --> 00:02:32,580 call stromatolites which appear fairly 53 00:02:39,770 --> 00:02:36,000 early on the earth and we also have 54 00:02:43,850 --> 00:02:39,780 other material repositories these stable 55 00:02:46,220 --> 00:02:43,860 isotopes and so light carbon like as 56 00:02:48,619 --> 00:02:46,230 shown here this is a reference to one of 57 00:02:50,990 --> 00:02:48,629 you you each rose papers that there's a 58 00:02:53,059 --> 00:02:51,000 sign of metabolism deep in the past 59 00:02:55,729 --> 00:02:53,069 that's apparently recorded by the 60 00:02:57,890 --> 00:02:55,739 distribution or fractionation of light 61 00:03:00,559 --> 00:02:57,900 and heavy carbon the different isotopes 62 00:03:02,870 --> 00:03:00,569 so other than these material phases that 63 00:03:06,740 --> 00:03:02,880 we can go out and find an old rocks we 64 00:03:09,380 --> 00:03:06,750 also have molecular biology and we've 65 00:03:10,970 --> 00:03:09,390 seen a couple of phylogenetic trees come 66 00:03:13,400 --> 00:03:10,980 up in the last couple days and a couple 67 00:03:15,979 --> 00:03:13,410 mentions of the concept of using 68 00:03:19,550 --> 00:03:15,989 molecular clocks to try to put a 69 00:03:21,680 --> 00:03:19,560 timeline on how DNA and protein 70 00:03:24,440 --> 00:03:21,690 sequences evolve through time so these 71 00:03:27,020 --> 00:03:24,450 are kind of two main repositories of 72 00:03:29,360 --> 00:03:27,030 biological knowledge and what I'm going 73 00:03:32,059 --> 00:03:29,370 to propose is a pathway to be able to 74 00:03:35,240 --> 00:03:32,069 link these two to linked sequence 75 00:03:36,680 --> 00:03:35,250 evolution of enzyme catalysis to actual 76 00:03:39,229 --> 00:03:36,690 repositories and I think that might help 77 00:03:43,250 --> 00:03:39,239 us calibrate things this is a long-term 78 00:03:45,680 --> 00:03:43,260 goal that I have here so the model 79 00:03:48,559 --> 00:03:45,690 system that I'm going to discuss today 80 00:03:50,420 --> 00:03:48,569 is microbial sulfate reduction these 81 00:03:52,220 --> 00:03:50,430 this way of thinking about things it's 82 00:03:54,259 --> 00:03:52,230 not limited at all to microbial sulfate 83 00:03:56,839 --> 00:03:54,269 reduction but microbial sulfate 84 00:04:00,289 --> 00:03:56,849 reduction is a as a convenient starter 85 00:04:02,420 --> 00:04:00,299 system for this way of thinking because 86 00:04:05,120 --> 00:04:02,430 there is a long history of measuring 87 00:04:07,490 --> 00:04:05,130 stable sulfur isotopes and their 88 00:04:11,390 --> 00:04:07,500 patterns of fractionation throughout 89 00:04:14,000 --> 00:04:11,400 geological history microbial sulfur 90 00:04:17,199 --> 00:04:14,010 sulfate reduction is a eight electron 91 00:04:19,879 --> 00:04:17,209 addition on the sulfate to make sulfide 92 00:04:22,339 --> 00:04:19,889 sulfate is pretty oxidizing molecule and 93 00:04:24,589 --> 00:04:22,349 so what these organisms are doing are 94 00:04:27,050 --> 00:04:24,599 taking electrons and using the energy 95 00:04:29,029 --> 00:04:27,060 released when those electrons move on to 96 00:04:32,330 --> 00:04:29,039 sulfates to power their growth 97 00:04:35,149 --> 00:04:32,340 metabolism and and it's and they're 98 00:04:36,439 --> 00:04:35,159 fairly diverse - I should say this is 99 00:04:38,390 --> 00:04:36,449 the net equation 100 00:04:40,219 --> 00:04:38,400 that we could write for this process we 101 00:04:41,869 --> 00:04:40,229 could calculate the energy of it think 102 00:04:43,730 --> 00:04:41,879 about the the cellular yields but 103 00:04:46,070 --> 00:04:43,740 they're they're quite diverse group of 104 00:04:49,610 --> 00:04:46,080 organisms that are all breathing or 105 00:04:51,950 --> 00:04:49,620 respiring sulfate and as I said there's 106 00:04:55,700 --> 00:04:51,960 a long tradition in geochemistry of 107 00:04:57,469 --> 00:04:55,710 measuring stable isotopes of sulfur 108 00:04:59,629 --> 00:04:57,479 through time and also in different 109 00:05:02,899 --> 00:04:59,639 environments and so what I'm showing 110 00:05:07,100 --> 00:05:02,909 here is a compilation of sulfur isotopes 111 00:05:09,709 --> 00:05:07,110 between sulfates and sulfide and we're 112 00:05:14,929 --> 00:05:09,719 looking at the two isotopes of sulfate 113 00:05:18,189 --> 00:05:14,939 of sulfur 32 and sulfur 34 and what is 114 00:05:21,829 --> 00:05:18,199 being shown here is in this gray area 115 00:05:26,209 --> 00:05:21,839 the equilibrium fractionation values of 116 00:05:28,010 --> 00:05:26,219 sulfate 2 sulfide for zero degrees 117 00:05:30,409 --> 00:05:28,020 Celsius all the way up to 40 degrees 118 00:05:32,719 --> 00:05:30,419 Celsius so the temperature at which 119 00:05:35,570 --> 00:05:32,729 sulfur isotopes will équilibre between 120 00:05:37,999 --> 00:05:35,580 sulfate and sulfide will affect the 121 00:05:40,249 --> 00:05:38,009 final value of the system and then we 122 00:05:42,529 --> 00:05:40,259 have all of this other stuff out here 123 00:05:45,290 --> 00:05:42,539 that I labeled kinetic this is the stuff 124 00:05:48,230 --> 00:05:45,300 that life does and so life is able to 125 00:05:51,260 --> 00:05:48,240 operate its metabolism in a way that's 126 00:05:53,600 --> 00:05:51,270 fast enough that sulfide is produced so 127 00:05:55,610 --> 00:05:53,610 fast that the isotopes aren't able to 128 00:05:58,519 --> 00:05:55,620 equilibrating so we get these deviations 129 00:06:01,610 --> 00:05:58,529 from equilibrated sulfur isotopes and 130 00:06:04,399 --> 00:06:01,620 these kinetic fractionation factors have 131 00:06:06,499 --> 00:06:04,409 been thought to be related to all sorts 132 00:06:08,989 --> 00:06:06,509 of stuff the rate of the organisms 133 00:06:11,360 --> 00:06:08,999 growth the place that they grow are they 134 00:06:12,860 --> 00:06:11,370 marine are they terrestrial are they 135 00:06:15,379 --> 00:06:12,870 pure culture are they are they mixed 136 00:06:17,540 --> 00:06:15,389 culture and it still remains an issue to 137 00:06:19,689 --> 00:06:17,550 define exactly which parameters are 138 00:06:24,379 --> 00:06:19,699 affecting the kinetic isotope 139 00:06:27,619 --> 00:06:24,389 fractionation values that life the 140 00:06:30,379 --> 00:06:27,629 result of biologic biology and why and 141 00:06:33,429 --> 00:06:30,389 which magnitude these isotopes are 142 00:06:36,949 --> 00:06:33,439 deviating away from the equilibrium if 143 00:06:39,829 --> 00:06:36,959 we spread those data along axis of time 144 00:06:43,239 --> 00:06:39,839 and we think about long ago here and 145 00:06:46,480 --> 00:06:43,249 today here and we look at sulfide 146 00:06:48,649 --> 00:06:46,490 isotopes compared to sulfate isotopes 147 00:06:50,430 --> 00:06:48,659 sulfide in the form of pyrite that's 148 00:06:51,870 --> 00:06:50,440 preserved we can see that 149 00:06:56,360 --> 00:06:51,880 in modern-day environments there's a 150 00:06:59,670 --> 00:06:56,370 huge spread of sulphate sulfide isotope 151 00:07:00,990 --> 00:06:59,680 fractionation and then in the past we 152 00:07:02,550 --> 00:07:01,000 have this problem that there's not a lot 153 00:07:05,940 --> 00:07:02,560 of data because there's not a lot of old 154 00:07:08,640 --> 00:07:05,950 material on the earth but the kind of 155 00:07:11,210 --> 00:07:08,650 variance here collapses and what I've 156 00:07:13,560 --> 00:07:11,220 marked on here is this 20 per mil 157 00:07:15,960 --> 00:07:13,570 fractionation value between sulfate and 158 00:07:17,460 --> 00:07:15,970 sulfide and so I'm going to ask the I'm 159 00:07:21,210 --> 00:07:17,470 going to try to address the question or 160 00:07:23,910 --> 00:07:21,220 speculate on why this 20% difference 161 00:07:25,650 --> 00:07:23,920 might be in the Archaean but in the in 162 00:07:28,260 --> 00:07:25,660 the present-day environment we get these 163 00:07:33,150 --> 00:07:28,270 very very large fracture nations in 164 00:07:36,450 --> 00:07:33,160 biology so what's going on in this 165 00:07:38,760 --> 00:07:36,460 process of sulfate reduction sulfate I 166 00:07:41,070 --> 00:07:38,770 mentioned before it's fairly oxidizing 167 00:07:43,860 --> 00:07:41,080 molecule you can put electrons on to it 168 00:07:45,810 --> 00:07:43,870 and when energy is released as electrons 169 00:07:47,670 --> 00:07:45,820 go on to sulfate gained energy from that 170 00:07:50,580 --> 00:07:47,680 process but it's actually chemically 171 00:07:53,370 --> 00:07:50,590 inert and what organisms have to do is 172 00:07:57,000 --> 00:07:53,380 they have to activate sulfate by using 173 00:07:59,700 --> 00:07:57,010 an ATP and in this activated form they 174 00:08:02,460 --> 00:07:59,710 are able to put first two electrons onto 175 00:08:06,060 --> 00:08:02,470 sulfate to make sulfite and then there's 176 00:08:08,790 --> 00:08:06,070 a six electron reduction of sulfite to 177 00:08:12,210 --> 00:08:08,800 make sulfide so this is the pathway or a 178 00:08:14,310 --> 00:08:12,220 very simplified version of microbial 179 00:08:16,470 --> 00:08:14,320 sulfate reduction the process of 180 00:08:18,690 --> 00:08:16,480 breathing on sulfate step one is to 181 00:08:20,640 --> 00:08:18,700 overcome this kinetic barrier and kind 182 00:08:23,100 --> 00:08:20,650 of power through that by using an ATP 183 00:08:25,230 --> 00:08:23,110 and the second step is a first two 184 00:08:27,810 --> 00:08:25,240 electron reduction and the third step is 185 00:08:31,710 --> 00:08:27,820 this pretty awesome I would I think six 186 00:08:34,980 --> 00:08:31,720 electron reduction of sulfite and for a 187 00:08:37,380 --> 00:08:34,990 long time people have recognized that 188 00:08:40,650 --> 00:08:37,390 there's a a pretty neat relationship 189 00:08:43,589 --> 00:08:40,660 between the ability or the extent of 190 00:08:46,950 --> 00:08:43,599 fractionation of sulfate sulfide 191 00:08:48,990 --> 00:08:46,960 isotopes proportional to the rate that 192 00:08:50,760 --> 00:08:49,000 these cells are growing so let's think 193 00:08:52,230 --> 00:08:50,770 let's explore this diagram a little bit 194 00:08:54,540 --> 00:08:52,240 it's a simple curve but it's a little 195 00:08:57,480 --> 00:08:54,550 bit tricky to think about I said before 196 00:09:00,870 --> 00:08:57,490 let me go back that the equilibrium 197 00:09:04,199 --> 00:09:00,880 value is essentially set by temperature 198 00:09:06,480 --> 00:09:04,209 over here and C's high values over 199 00:09:09,299 --> 00:09:06,490 here this is around 60 per mill 80 per 200 00:09:10,859 --> 00:09:09,309 mill here and what biology is doing is 201 00:09:14,309 --> 00:09:10,869 spreading the data away from that 202 00:09:16,379 --> 00:09:14,319 equilibrium fractionation this is 203 00:09:18,239 --> 00:09:16,389 another way of drawing that but it's a 204 00:09:21,239 --> 00:09:18,249 way of drying it that emphasizes a rate 205 00:09:23,639 --> 00:09:21,249 dependence on the process here we're 206 00:09:24,989 --> 00:09:23,649 talking about fractionation factors in 207 00:09:26,850 --> 00:09:24,999 an inverse way so this is a little bit 208 00:09:28,679 --> 00:09:26,860 confusing but we could translate this 209 00:09:30,389 --> 00:09:28,689 number here to about a sixty per mil 210 00:09:33,720 --> 00:09:30,399 fractionation that's that equilibrium 211 00:09:36,509 --> 00:09:33,730 value that would be met as the rate goes 212 00:09:38,579 --> 00:09:36,519 to zero we would encounter the y-axis 213 00:09:40,470 --> 00:09:38,589 here and that would happen depending on 214 00:09:43,290 --> 00:09:40,480 the temperature right around 60 or 280 215 00:09:46,400 --> 00:09:43,300 per ml fractionation but as cells grow 216 00:09:49,109 --> 00:09:46,410 faster and faster the fractionation 217 00:09:51,329 --> 00:09:49,119 changes it's kind of muted away from 218 00:09:54,059 --> 00:09:51,339 that equilibrium value this is an old 219 00:09:56,189 --> 00:09:54,069 observation that has been reproduced 220 00:09:58,739 --> 00:09:56,199 again and again historically about every 221 00:10:01,319 --> 00:09:58,749 10 years actually another group is able 222 00:10:02,790 --> 00:10:01,329 to reproduce this curve and it leads to 223 00:10:06,840 --> 00:10:02,800 this question of like why does why does 224 00:10:07,650 --> 00:10:06,850 this happen and a couple years ago I 225 00:10:10,530 --> 00:10:07,660 think there was a pretty nice 226 00:10:12,059 --> 00:10:10,540 breakthrough in my mind with trying to 227 00:10:14,929 --> 00:10:12,069 understand the shape of this curve and 228 00:10:17,579 --> 00:10:14,939 that breakthrough was accomplished in a 229 00:10:19,730 --> 00:10:17,589 model that related the rate of the 230 00:10:22,109 --> 00:10:19,740 process the energy of the process and 231 00:10:23,549 --> 00:10:22,119 knowledge of the fractionation factors 232 00:10:25,590 --> 00:10:23,559 of these enzymes or the inferred 233 00:10:28,230 --> 00:10:25,600 fractionation factors of these enzymes 234 00:10:30,509 --> 00:10:28,240 and really this is coming out of some 235 00:10:32,429 --> 00:10:30,519 work a while ago by Clinton stoner 236 00:10:35,340 --> 00:10:32,439 and which was later on followed up by 237 00:10:37,699 --> 00:10:35,350 Dan beard and honking I don't know how 238 00:10:42,799 --> 00:10:37,709 to say that name I shouldn't try sorry 239 00:10:45,689 --> 00:10:42,809 these folks of trying to relate 240 00:10:49,410 --> 00:10:45,699 reversibility of a chemical process to 241 00:10:51,359 --> 00:10:49,420 isotope BAC flux and the the force or 242 00:10:56,009 --> 00:10:51,369 the chemical potential of a given 243 00:10:58,289 --> 00:10:56,019 reaction and so this knowledge of being 244 00:11:00,660 --> 00:10:58,299 able to relate the chemical potential of 245 00:11:02,999 --> 00:11:00,670 a reaction to the BAC flux which is 246 00:11:06,720 --> 00:11:03,009 related to isotope exchange was employed 247 00:11:09,509 --> 00:11:06,730 by Baz weighing and eat I have Lee in a 248 00:11:11,249 --> 00:11:09,519 2014 paper were they yours 249 00:11:13,499 --> 00:11:11,259 they were able to put a line on this 250 00:11:15,539 --> 00:11:13,509 data by building a model and so they 251 00:11:17,669 --> 00:11:15,549 could say okay now we have a model that 252 00:11:17,940 --> 00:11:17,679 describes the state of a cell we know 253 00:11:19,920 --> 00:11:17,950 that 254 00:11:21,810 --> 00:11:19,930 parameters of the model and we can we 255 00:11:24,350 --> 00:11:21,820 can make a line that actually fits the 256 00:11:26,960 --> 00:11:24,360 data and so this is very very satisfying 257 00:11:29,970 --> 00:11:26,970 because what they were able to do is 258 00:11:32,160 --> 00:11:29,980 infer the processes that are occurring 259 00:11:34,860 --> 00:11:32,170 inside of a cell here's a cell that's 260 00:11:37,110 --> 00:11:34,870 doing microbial sulfate reduction and 261 00:11:39,270 --> 00:11:37,120 they broke the process apart and put 262 00:11:41,430 --> 00:11:39,280 those components into their model which 263 00:11:44,610 --> 00:11:41,440 is relying on knowledge of rate and 264 00:11:47,310 --> 00:11:44,620 isotope fractionation to say when 265 00:11:48,990 --> 00:11:47,320 sulfate is imported into the cell there 266 00:11:51,510 --> 00:11:49,000 might be a fractionation that occurs and 267 00:11:54,390 --> 00:11:51,520 then here's our activation step this is 268 00:11:57,330 --> 00:11:54,400 to get get rid of this kinetic problem 269 00:12:00,870 --> 00:11:57,340 here's the this third step the first two 270 00:12:03,060 --> 00:12:00,880 electron reduction of of the sulfur atom 271 00:12:05,130 --> 00:12:03,070 and then there's this final step there's 272 00:12:09,720 --> 00:12:05,140 six electron reduction so this seems 273 00:12:11,070 --> 00:12:09,730 like a satisfying way of looking at 274 00:12:15,750 --> 00:12:11,080 things to be able to understand the 275 00:12:17,430 --> 00:12:15,760 shape of that curve but it left us still 276 00:12:19,740 --> 00:12:17,440 with a little bit of a missing some 277 00:12:20,760 --> 00:12:19,750 missing data we could still take the 278 00:12:23,580 --> 00:12:20,770 model and come up with different 279 00:12:26,040 --> 00:12:23,590 scenarios to accomplish the same isotope 280 00:12:29,000 --> 00:12:26,050 fractionation another this is a common 281 00:12:31,140 --> 00:12:29,010 process in science many hypotheses can 282 00:12:33,330 --> 00:12:31,150 accomplice aim data set so there's a 283 00:12:36,900 --> 00:12:33,340 degeneracy built into this model and 284 00:12:39,930 --> 00:12:36,910 that degeneracy I'm seeking to get rid 285 00:12:42,780 --> 00:12:39,940 of by trying to find out two pieces of 286 00:12:45,270 --> 00:12:42,790 knowledge that have not really been 287 00:12:47,280 --> 00:12:45,280 described one what are the actual 288 00:12:48,720 --> 00:12:47,290 fractionation factors that are happening 289 00:12:50,490 --> 00:12:48,730 at the different steps in the metabolism 290 00:12:53,070 --> 00:12:50,500 these have never been determined 291 00:12:57,300 --> 00:12:53,080 empirically and instead they've just 292 00:12:58,680 --> 00:12:57,310 been estimated numerically and also what 293 00:13:00,450 --> 00:12:58,690 is the rate determining step of the 294 00:13:02,280 --> 00:13:00,460 metabolism knowledge of the rate 295 00:13:03,930 --> 00:13:02,290 determining step of metabolism is very 296 00:13:05,190 --> 00:13:03,940 very important in this case because 297 00:13:07,620 --> 00:13:05,200 that's going to be the bottleneck that 298 00:13:09,780 --> 00:13:07,630 will be expressed that'll be the 299 00:13:12,420 --> 00:13:09,790 fractionation factor that we actually 300 00:13:14,750 --> 00:13:12,430 measure later on it will be dependent on 301 00:13:18,060 --> 00:13:14,760 that rate determining determining step 302 00:13:22,050 --> 00:13:18,070 so to try to determine what that rate 303 00:13:24,780 --> 00:13:22,060 limiting step is or in part to do that 304 00:13:26,220 --> 00:13:24,790 min sub was actually able to make some 305 00:13:28,710 --> 00:13:26,230 measurements of intracellular 306 00:13:31,079 --> 00:13:28,720 concentrations of these sulfur and 307 00:13:33,179 --> 00:13:31,089 metabolites in the cell and 308 00:13:35,309 --> 00:13:33,189 here's a complicated graph and min sub 309 00:13:37,829 --> 00:13:35,319 uses kind of funny symbols so be careful 310 00:13:39,689 --> 00:13:37,839 here here is cell growth here we're 311 00:13:41,669 --> 00:13:39,699 monitoring the production of sulfide 312 00:13:45,289 --> 00:13:41,679 it's the respiratory product of the cell 313 00:13:47,399 --> 00:13:45,299 so the cells grow sulfate Goes Down and 314 00:13:49,349 --> 00:13:47,409 sulfates inside of the cell kind of 315 00:13:51,359 --> 00:13:49,359 bounces around in concentration it's not 316 00:13:52,739 --> 00:13:51,369 clear really what it's related to up 317 00:13:55,529 --> 00:13:52,749 here but it's pretty high in the cell 318 00:13:56,729 --> 00:13:55,539 then here's the concentration of ApS in 319 00:13:58,589 --> 00:13:56,739 the cell this is the kinetically 320 00:14:00,479 --> 00:13:58,599 activated sulfur compound that those 321 00:14:02,549 --> 00:14:00,489 first two electrons will be put onto and 322 00:14:05,399 --> 00:14:02,559 then here's the sulfite this is the 323 00:14:07,799 --> 00:14:05,409 compound that has six electrons added on 324 00:14:09,779 --> 00:14:07,809 to it and what min sub found was that 325 00:14:13,919 --> 00:14:09,789 basically that ApS is always at a higher 326 00:14:15,649 --> 00:14:13,929 concentration than sulfite and so one 327 00:14:18,659 --> 00:14:15,659 thing this this implies is that 328 00:14:20,729 --> 00:14:18,669 kinetically this this aps reduction step 329 00:14:22,859 --> 00:14:20,739 is a little bit slower than the sulfite 330 00:14:24,419 --> 00:14:22,869 step and once sulphide is produced the 331 00:14:27,299 --> 00:14:24,429 final six electrons go right onto 332 00:14:28,979 --> 00:14:27,309 sulfite making sulfide and this this 333 00:14:31,979 --> 00:14:28,989 seems fairly comfortable chemically 334 00:14:34,739 --> 00:14:31,989 sulfites pretty pretty reactive and so 335 00:14:36,599 --> 00:14:34,749 it kind of goes to this the suggestion 336 00:14:38,969 --> 00:14:36,609 that it may be this ApS step is rate 337 00:14:40,919 --> 00:14:38,979 limiting if that's the case this ApS 338 00:14:42,659 --> 00:14:40,929 step will be very important in 339 00:14:44,609 --> 00:14:42,669 controlling the observed sulphur isotope 340 00:14:47,669 --> 00:14:44,619 fraction fractionation factor of the 341 00:14:50,909 --> 00:14:47,679 metabolism and so we could say that 342 00:14:52,349 --> 00:14:50,919 possibly this rate determining step or 343 00:14:54,359 --> 00:14:52,359 at least in some cellular conditions 344 00:14:57,119 --> 00:14:54,369 would be that first two electron 345 00:15:00,059 --> 00:14:57,129 reduction of ApS and that leaves us with 346 00:15:02,129 --> 00:15:00,069 a second part missing part of the data 347 00:15:06,209 --> 00:15:02,139 here is the actual numbers that are 348 00:15:08,369 --> 00:15:06,219 associated with these enzymes as an 349 00:15:10,169 --> 00:15:08,379 individual catalyst what is the isotopes 350 00:15:13,109 --> 00:15:10,179 that stable isotope fractionation of 351 00:15:17,069 --> 00:15:13,119 this catalyst so we we sought to address 352 00:15:21,479 --> 00:15:17,079 that question by purifying the enzyme 353 00:15:23,159 --> 00:15:21,489 and a say in it using isotope ratio a 354 00:15:24,809 --> 00:15:23,169 mass spectrometry of the substrates and 355 00:15:27,089 --> 00:15:24,819 products to determine that value so 356 00:15:28,979 --> 00:15:27,099 here's the enzyme remember it takes this 357 00:15:30,899 --> 00:15:28,989 kinetically activated form of sulfate 358 00:15:34,139 --> 00:15:30,909 and adds two electrons on to it to make 359 00:15:36,389 --> 00:15:34,149 sulfite here are some other iron sulfur 360 00:15:38,099 --> 00:15:36,399 clusters that are found in biology this 361 00:15:40,199 --> 00:15:38,109 time these iron sulfur clusters are 362 00:15:43,039 --> 00:15:40,209 relatively high potential they're not 363 00:15:44,730 --> 00:15:43,049 sitting at minus 500 millivolts 364 00:15:48,210 --> 00:15:44,740 ferredoxin or more 365 00:15:49,980 --> 00:15:48,220 than that and that's because sulfate is 366 00:15:51,720 --> 00:15:49,990 more oxidizing than that you could 367 00:15:53,579 --> 00:15:51,730 imagine electrons coming in to these 368 00:15:55,560 --> 00:15:53,589 four iron four sulfur Q Bane's and then 369 00:15:58,079 --> 00:15:55,570 being transferred on to this fa D which 370 00:16:01,380 --> 00:15:58,089 is hiding up here in yellow and then at 371 00:16:02,460 --> 00:16:01,390 that stage sulfite is going to dock 372 00:16:04,199 --> 00:16:02,470 somewhere around here 373 00:16:06,210 --> 00:16:04,209 we're sorry the APS is going to dock 374 00:16:12,930 --> 00:16:06,220 around here and sulfite will be produced 375 00:16:16,070 --> 00:16:12,940 so he D akio gotta who was previously at 376 00:16:20,639 --> 00:16:16,080 the NPI for chemical energy conversion 377 00:16:23,190 --> 00:16:20,649 he grows you know 200 liter vats of this 378 00:16:25,440 --> 00:16:23,200 organism dissolve of Vibrio Voges and so 379 00:16:27,660 --> 00:16:25,450 he was able to donate some of this 380 00:16:29,340 --> 00:16:27,670 protein material to the laboratory and 381 00:16:32,670 --> 00:16:29,350 save us a lot of work he did a partial 382 00:16:36,510 --> 00:16:32,680 purification of the APR a and B sub 383 00:16:39,600 --> 00:16:36,520 units and sent them to us at which time 384 00:16:43,170 --> 00:16:39,610 min sub was able to conduct protein 385 00:16:46,860 --> 00:16:43,180 assays and what he did was he put the 386 00:16:49,170 --> 00:16:46,870 substrate of the enzyme the enzyme and 387 00:16:51,930 --> 00:16:49,180 an artificial electron donor into a tube 388 00:16:53,940 --> 00:16:51,940 in an anaerobic environment and then he 389 00:16:56,760 --> 00:16:53,950 was able to use ion chromatography to 390 00:16:58,500 --> 00:16:56,770 separate out the products and the 391 00:17:01,079 --> 00:16:58,510 substrate of this reaction so in this 392 00:17:03,060 --> 00:17:01,089 case he's going to collect a PS the 393 00:17:05,069 --> 00:17:03,070 substrate of the reaction and sulfite 394 00:17:09,540 --> 00:17:05,079 the product of the reaction and then 395 00:17:11,850 --> 00:17:09,550 later on take those molecules and launch 396 00:17:14,689 --> 00:17:11,860 them into the Neptune instrument and 397 00:17:18,329 --> 00:17:14,699 we'll be able to determine the 3234 398 00:17:20,579 --> 00:17:18,339 isotope values in a compound specific 399 00:17:23,699 --> 00:17:20,589 way and be able to understand isotope 400 00:17:25,910 --> 00:17:23,709 fractionation value of the enzyme here 401 00:17:29,010 --> 00:17:25,920 are the enzyme kinetics at two different 402 00:17:31,560 --> 00:17:29,020 temperatures and so what min sub was 403 00:17:34,200 --> 00:17:31,570 able to show was that cold slows the 404 00:17:37,040 --> 00:17:34,210 enzyme down and heat speeds it up so 405 00:17:39,930 --> 00:17:37,050 change of 12 degrees changed the rate of 406 00:17:42,450 --> 00:17:39,940 sulfite production by around five times 407 00:17:45,419 --> 00:17:42,460 a pretty dramatic change in the rate of 408 00:17:51,270 --> 00:17:45,429 the enzyme and he was able to monitor 409 00:17:54,000 --> 00:17:51,280 the sulfur isotope compound value for 410 00:17:55,770 --> 00:17:54,010 aps the substrate of the reaction and 411 00:17:57,900 --> 00:17:55,780 the product of the reaction and then 412 00:17:58,649 --> 00:17:57,910 make these basically Rayleigh 413 00:18:01,200 --> 00:17:58,659 fractionation 414 00:18:06,089 --> 00:18:01,210 and curves where the slope of this line 415 00:18:07,680 --> 00:18:06,099 is indicative of the 3432 sulphur 416 00:18:10,710 --> 00:18:07,690 isotope difference between the product 417 00:18:12,359 --> 00:18:10,720 and the reaction reactant here I said 418 00:18:16,379 --> 00:18:12,369 before that the rate of the enzyme here 419 00:18:19,229 --> 00:18:16,389 is 5 times faster than the cool 20 420 00:18:21,029 --> 00:18:19,239 degree assay version here but something 421 00:18:22,830 --> 00:18:21,039 interesting the isotope fractionation 422 00:18:27,080 --> 00:18:22,840 factor is basically the same between 423 00:18:30,109 --> 00:18:27,090 these two cases and so with this in hand 424 00:18:33,629 --> 00:18:30,119 we have a model that describes 425 00:18:35,849 --> 00:18:33,639 mathematically how isotopes will will be 426 00:18:39,299 --> 00:18:35,859 partitioned from sulfate to sulfide 427 00:18:40,499 --> 00:18:39,309 under various cellular states and now we 428 00:18:43,049 --> 00:18:40,509 have a real number 429 00:18:46,619 --> 00:18:43,059 now the first number of a purified 430 00:18:53,070 --> 00:18:46,629 enzyme I should qualify that statement 431 00:18:54,629 --> 00:18:53,080 by saying entirely pure non missing 432 00:18:56,729 --> 00:18:54,639 subunit version of the enzyme here 433 00:18:58,529 --> 00:18:56,739 previously about two years ago 434 00:18:59,909 --> 00:18:58,539 another group measured an enzyme but it 435 00:19:02,759 --> 00:18:59,919 was missing a subunit and so this was 436 00:19:04,859 --> 00:19:02,769 actually the first purified complete 437 00:19:07,619 --> 00:19:04,869 enzyme component for which sulfur 438 00:19:10,680 --> 00:19:07,629 isotope values have been measured and we 439 00:19:13,409 --> 00:19:10,690 also have this data before which is 440 00:19:16,469 --> 00:19:13,419 suggestive of this rate limiting step of 441 00:19:18,330 --> 00:19:16,479 this enzymes enzyme here and so what we 442 00:19:22,289 --> 00:19:18,340 did is we took this previously 443 00:19:24,629 --> 00:19:22,299 constructed model and we started 444 00:19:27,269 --> 00:19:24,639 plotting expected isotope fractionation 445 00:19:29,159 --> 00:19:27,279 values of the cells for different 446 00:19:30,839 --> 00:19:29,169 chemical conditions different chemical 447 00:19:33,930 --> 00:19:30,849 conditions in terms of the driving force 448 00:19:36,239 --> 00:19:33,940 of the metabolic reaction so if the 449 00:19:38,820 --> 00:19:36,249 cells have lots of energy that would be 450 00:19:40,889 --> 00:19:38,830 sitting over here at these more negative 451 00:19:43,229 --> 00:19:40,899 redox potentials and if the cells have 452 00:19:45,749 --> 00:19:43,239 less energy they would be using more 453 00:19:47,519 --> 00:19:45,759 oxidizing electron donors or higher 454 00:19:49,889 --> 00:19:47,529 potential electron donors to review 455 00:19:52,349 --> 00:19:49,899 sulfate and they would have less driving 456 00:19:53,969 --> 00:19:52,359 force behind their reaction and what we 457 00:19:56,399 --> 00:19:53,979 found when we started plot in the data 458 00:19:59,009 --> 00:19:56,409 like this in this model was that 459 00:20:01,739 --> 00:19:59,019 actually the observed isotope 460 00:20:03,989 --> 00:20:01,749 fractionation of the cell does not ever 461 00:20:07,829 --> 00:20:03,999 go over the isotope fractionation of 462 00:20:09,930 --> 00:20:07,839 this one single enzyme until that enzyme 463 00:20:11,430 --> 00:20:09,940 step becomes reversible and so this is 464 00:20:14,799 --> 00:20:11,440 this BAC flux 465 00:20:17,259 --> 00:20:14,809 a contribution here when there's not a 466 00:20:19,899 --> 00:20:17,269 sufficient amount of energy to push that 467 00:20:22,930 --> 00:20:19,909 reaction forward the isotopes can react 468 00:20:24,909 --> 00:20:22,940 Willa Breit across between APs and 469 00:20:27,729 --> 00:20:24,919 sulfite and that's what leads to these 470 00:20:30,279 --> 00:20:27,739 large close to equilibrium fraction 471 00:20:33,129 --> 00:20:30,289 nations and we're able to do that for a 472 00:20:35,379 --> 00:20:33,139 number of different rates of microbial 473 00:20:36,639 --> 00:20:35,389 sulfate reduction and you can see when 474 00:20:38,769 --> 00:20:36,649 cells are really cranking through 475 00:20:41,229 --> 00:20:38,779 sulfate and making sulfide that kind of 476 00:20:43,449 --> 00:20:41,239 mutes everything they're pulling the 477 00:20:45,909 --> 00:20:43,459 sulfate through their metabolism so fast 478 00:20:48,639 --> 00:20:45,919 that it doesn't ever have time to 479 00:20:51,669 --> 00:20:48,649 approach these equilibrium values but at 480 00:20:53,469 --> 00:20:51,679 these slower rates again the situation 481 00:20:56,829 --> 00:20:53,479 as long as the cells have sufficient 482 00:20:59,049 --> 00:20:56,839 electron driving force the sulfur 483 00:21:01,810 --> 00:20:59,059 isotopes can't really ever go above this 484 00:21:05,560 --> 00:21:01,820 value which is set kinetically on this 485 00:21:07,839 --> 00:21:05,570 one particular enzyme so that's just 486 00:21:11,259 --> 00:21:07,849 what I said it can't increase more than 487 00:21:14,680 --> 00:21:11,269 the actual aps enzyme step when there's 488 00:21:16,180 --> 00:21:14,690 sufficient energy so that's kind of the 489 00:21:17,919 --> 00:21:16,190 nuts and bolts of the data that I want 490 00:21:20,049 --> 00:21:17,929 to present and I want to kind of 491 00:21:23,349 --> 00:21:20,059 hypothesize about the possible 492 00:21:26,709 --> 00:21:23,359 implications of this purified enzyme 493 00:21:28,680 --> 00:21:26,719 sulfur isotope values as it might have 494 00:21:30,729 --> 00:21:28,690 something to do with our historical 495 00:21:32,949 --> 00:21:30,739 interpretation of these isotope values 496 00:21:35,499 --> 00:21:32,959 and so we can ask this question why in 497 00:21:36,819 --> 00:21:35,509 the Archaean or do we not see close to 498 00:21:38,769 --> 00:21:36,829 equilibrium sulphur isotope 499 00:21:41,739 --> 00:21:38,779 fractionation values here we see these 500 00:21:43,239 --> 00:21:41,749 really this really big spread some of 501 00:21:45,849 --> 00:21:43,249 them are narrow some of but there's a 502 00:21:49,269 --> 00:21:45,859 lot of variance here and one thing we 503 00:21:51,129 --> 00:21:49,279 might say is that in the modern 504 00:21:53,799 --> 00:21:51,139 environment there there's a huge 505 00:21:56,440 --> 00:21:53,809 diversity of environments and but in 506 00:21:58,239 --> 00:21:56,450 particular these organisms are under a 507 00:22:00,940 --> 00:21:58,249 lot of competition and they might be 508 00:22:03,249 --> 00:22:00,950 using very modest electron donors and 509 00:22:05,109 --> 00:22:03,259 there's very modest electron donors put 510 00:22:06,789 --> 00:22:05,119 us over over here on the right side of 511 00:22:10,049 --> 00:22:06,799 our graph where the cells are growing 512 00:22:12,279 --> 00:22:10,059 closer to equilibrium and therefore 513 00:22:15,729 --> 00:22:12,289 relating these larger isotope 514 00:22:19,449 --> 00:22:15,739 fractionation values it's very tempting 515 00:22:21,729 --> 00:22:19,459 to speculate that a lot using the same 516 00:22:25,180 --> 00:22:21,739 similar line of thinking that in the 517 00:22:27,460 --> 00:22:25,190 Archaean the fact that we don't see 518 00:22:29,830 --> 00:22:27,470 large sulphur isotope fractionation x' 519 00:22:33,010 --> 00:22:29,840 might be coincident with the idea that 520 00:22:35,140 --> 00:22:33,020 these organisms were electron replete 521 00:22:37,300 --> 00:22:35,150 they had a lot of power to run their 522 00:22:42,940 --> 00:22:37,310 metabolism and they essentially weren't 523 00:22:44,740 --> 00:22:42,950 starving for electrons and to help help 524 00:22:47,260 --> 00:22:44,750 us kind of probe around this idea and 525 00:22:49,710 --> 00:22:47,270 interpret this idea we can think of what 526 00:22:52,300 --> 00:22:49,720 sulfate reduction might have meant 527 00:22:54,940 --> 00:22:52,310 evolutionarily if you were a microbe 528 00:22:57,580 --> 00:22:54,950 swimming in the ocean in the Archaean 529 00:22:59,680 --> 00:22:57,590 and you were using hydrogen if you were 530 00:23:01,840 --> 00:22:59,690 a sulfate reducer or methanogens or an 531 00:23:04,990 --> 00:23:01,850 acid again we could use the concept of 532 00:23:08,500 --> 00:23:05,000 the threshold concentration of hydrogen 533 00:23:11,110 --> 00:23:08,510 and the expected energy yield for the 534 00:23:14,080 --> 00:23:11,120 same concentration of hydrogen to either 535 00:23:15,700 --> 00:23:14,090 take electrons from hydrogen and reduce 536 00:23:17,710 --> 00:23:15,710 sulfate or take electrons from hydrogen 537 00:23:20,110 --> 00:23:17,720 and reduce co2 in either of these two 538 00:23:22,030 --> 00:23:20,120 different ways and because sulfate is 539 00:23:24,310 --> 00:23:22,040 such a good electron acceptor because as 540 00:23:27,130 --> 00:23:24,320 a more positive midpoint potential it's 541 00:23:28,990 --> 00:23:27,140 easier to reduce than co2 what sulphur 542 00:23:30,670 --> 00:23:29,000 sulfate reducers are able to do and 543 00:23:32,530 --> 00:23:30,680 people have known about this for a long 544 00:23:34,330 --> 00:23:32,540 time they're able to suck hydrogen 545 00:23:37,240 --> 00:23:34,340 concentrations down to a much lower 546 00:23:39,100 --> 00:23:37,250 level than either of these organisms I 547 00:23:41,350 --> 00:23:39,110 think a good way to interpret this is 548 00:23:43,690 --> 00:23:41,360 just to look at these Gibbs free energy 549 00:23:47,250 --> 00:23:43,700 of reactions for the electron addition 550 00:23:49,570 --> 00:23:47,260 on to either of these acceptors and so 551 00:23:52,270 --> 00:23:49,580 probably the evolution of sulfate 552 00:23:54,130 --> 00:23:52,280 reduction was a major bioenergetic 553 00:23:56,410 --> 00:23:54,140 innovation and what that allowed these 554 00:23:58,180 --> 00:23:56,420 organisms to do is in the case of using 555 00:24:00,670 --> 00:23:58,190 a common electron donor or was they were 556 00:24:02,350 --> 00:24:00,680 able to out-compete anything else that 557 00:24:05,140 --> 00:24:02,360 was living around they're putting 558 00:24:07,780 --> 00:24:05,150 electrons onto very meager electron 559 00:24:09,550 --> 00:24:07,790 acceptors like co2 and that would be 560 00:24:11,850 --> 00:24:09,560 kind of coincident coincident with this 561 00:24:15,310 --> 00:24:11,860 idea that they were running electron 562 00:24:17,470 --> 00:24:15,320 replete metabolisms after you know today 563 00:24:19,750 --> 00:24:17,480 these organisms are shoved into all 564 00:24:22,360 --> 00:24:19,760 sorts of different energy environments 565 00:24:25,660 --> 00:24:22,370 and they're living off of a lot of 566 00:24:27,430 --> 00:24:25,670 scraps where they they don't have a lot 567 00:24:29,710 --> 00:24:27,440 of driving force in their reaction and 568 00:24:31,750 --> 00:24:29,720 that might be one way to interpret this 569 00:24:33,760 --> 00:24:31,760 historical distribution of stable 570 00:24:35,350 --> 00:24:33,770 isotopes through time very replete 571 00:24:37,990 --> 00:24:35,360 conditions lots of electron donor 572 00:24:38,690 --> 00:24:38,000 they're competing against things with 573 00:24:40,730 --> 00:24:38,700 very 574 00:24:42,830 --> 00:24:40,740 artists electron acceptors but today 575 00:24:44,270 --> 00:24:42,840 there's a lot more competition there's a 576 00:24:50,060 --> 00:24:44,280 lot more high potential metabolisms 577 00:24:51,860 --> 00:24:50,070 available and I want to take this 578 00:24:53,690 --> 00:24:51,870 concept a little bit further and spit 579 00:24:56,540 --> 00:24:53,700 and use use our knowledge of isotopes 580 00:24:58,370 --> 00:24:56,550 here to speculate on processes of energy 581 00:25:01,280 --> 00:24:58,380 conservation that might be occurring in 582 00:25:03,560 --> 00:25:01,290 these cells I said earlier that these 583 00:25:06,460 --> 00:25:03,570 microbes are quite metabolically diverse 584 00:25:10,130 --> 00:25:06,470 and they are and in fact we don't have a 585 00:25:11,960 --> 00:25:10,140 unified understanding of how electron 586 00:25:13,550 --> 00:25:11,970 transfer processes are coupled to energy 587 00:25:16,340 --> 00:25:13,560 conservation in these cells or energy 588 00:25:18,200 --> 00:25:16,350 conversion if you will to say that 589 00:25:19,460 --> 00:25:18,210 simply we don't know which steps in 590 00:25:22,450 --> 00:25:19,470 these metabolisms are coupled to 591 00:25:25,400 --> 00:25:22,460 chemiosmotic potential generation and 592 00:25:27,020 --> 00:25:25,410 that will probably be variable for 593 00:25:29,000 --> 00:25:27,030 different types of cells but it's 594 00:25:30,800 --> 00:25:29,010 interesting for me to consider whether 595 00:25:33,440 --> 00:25:30,810 or not we could use isotopes as a way of 596 00:25:35,870 --> 00:25:33,450 rule of making hypotheses that are 597 00:25:37,490 --> 00:25:35,880 testable here so here's a picture of a 598 00:25:40,400 --> 00:25:37,500 cell that might be putting electrons 599 00:25:42,140 --> 00:25:40,410 onto sulfate pumping ions out in that 600 00:25:43,730 --> 00:25:42,150 process and then making ATP as those 601 00:25:46,610 --> 00:25:43,740 ions come back in across the potential 602 00:25:49,790 --> 00:25:46,620 and we could think about growing a 603 00:25:51,560 --> 00:25:49,800 sulphate reducer in a laboratory 604 00:25:53,570 --> 00:25:51,570 environment this is something that I 605 00:25:56,270 --> 00:25:53,580 would like to do or if you want to do it 606 00:25:58,190 --> 00:25:56,280 please talk to me and we can do it 607 00:25:59,780 --> 00:25:58,200 together you have some sulfate reducers 608 00:26:02,090 --> 00:25:59,790 growing at home but you could imagine 609 00:26:05,090 --> 00:26:02,100 growing sulfate reducers with a chemical 610 00:26:08,630 --> 00:26:05,100 potential for their metabolism and you 611 00:26:10,670 --> 00:26:08,640 could set them by modulating the 612 00:26:11,870 --> 00:26:10,680 concentrations and the temperature and 613 00:26:14,150 --> 00:26:11,880 you could grow them at this potential 614 00:26:17,000 --> 00:26:14,160 and you could say well if this organism 615 00:26:18,050 --> 00:26:17,010 was pushing against a membrane bound ion 616 00:26:19,430 --> 00:26:18,060 potential of a hundred and eighty 617 00:26:21,800 --> 00:26:19,440 millivolts that would translate into 618 00:26:23,800 --> 00:26:21,810 about 20 kilojoules per mole and then 619 00:26:27,320 --> 00:26:23,810 you would expect the cells to be able to 620 00:26:28,880 --> 00:26:27,330 gain or to have a free or remaining 621 00:26:30,920 --> 00:26:28,890 amount of energy of about 10 kilojoules 622 00:26:33,230 --> 00:26:30,930 per mole of reaction this would mean 623 00:26:35,120 --> 00:26:33,240 more reversible and more fractionation 624 00:26:37,310 --> 00:26:35,130 if it was coupled but if you took those 625 00:26:38,780 --> 00:26:37,320 same cells and they weren't coupled they 626 00:26:40,070 --> 00:26:38,790 weren't pushing against the ion 627 00:26:42,170 --> 00:26:40,080 potential they weren't using this to 628 00:26:44,150 --> 00:26:42,180 direct directly conserve energy you 629 00:26:47,420 --> 00:26:44,160 would get zero minus 30 so minus minus 630 00:26:50,420 --> 00:26:47,430 thirty kilovolts for a reaction and from 631 00:26:52,250 --> 00:26:50,430 a sulphur isotope perspective what this 632 00:26:54,020 --> 00:26:52,260 would mean would it be a 633 00:26:55,640 --> 00:26:54,030 where it's less reversible and less 634 00:26:57,470 --> 00:26:55,650 fractionation this is a testable 635 00:27:00,020 --> 00:26:57,480 hypothesis that we might be able to use 636 00:27:01,580 --> 00:27:00,030 isotope fractionation from whole cell 637 00:27:03,310 --> 00:27:01,590 environments to be able to find 638 00:27:09,200 --> 00:27:03,320 something out about their biophysical an 639 00:27:13,130 --> 00:27:09,210 energy transduction system so the next 640 00:27:15,860 --> 00:27:13,140 steps that were pretty excited to do now 641 00:27:18,020 --> 00:27:15,870 that we have one data point on one of 642 00:27:19,850 --> 00:27:18,030 these enzymes is to try to entertain 643 00:27:23,840 --> 00:27:19,860 this possibility of looking across the 644 00:27:25,220 --> 00:27:23,850 sequence phylogeny of these see these 645 00:27:26,840 --> 00:27:25,230 sequences are found in different 646 00:27:28,160 --> 00:27:26,850 organisms these different homologues and 647 00:27:30,440 --> 00:27:28,170 see if there's any evolutionary 648 00:27:32,480 --> 00:27:30,450 variation on this catalyst through time 649 00:27:34,490 --> 00:27:32,490 and I think that will be very very 650 00:27:36,820 --> 00:27:34,500 important for us to understand whether 651 00:27:39,590 --> 00:27:36,830 or not this hypothesis that I set up it 652 00:27:41,870 --> 00:27:39,600 could be true at all if there's a lot of 653 00:27:45,380 --> 00:27:41,880 isotope variability certainly we can't 654 00:27:49,220 --> 00:27:45,390 go and make claims about how this - 20 655 00:27:51,080 --> 00:27:49,230 per mil number has meaning in deep time 656 00:27:54,860 --> 00:27:51,090 instead we'll be left with a question of 657 00:27:57,350 --> 00:27:54,870 what are the ancestral state variants of 658 00:27:58,670 --> 00:27:57,360 these enzymes operating out in terms of 659 00:27:59,990 --> 00:27:58,680 their sulfur isotopes and so that's 660 00:28:02,720 --> 00:28:00,000 something that we'll do in the future is 661 00:28:04,880 --> 00:28:02,730 try to gain some evolutionary insight 662 00:28:06,500 --> 00:28:04,890 onto the history of sulfur isotope 663 00:28:09,350 --> 00:28:06,510 fractionation from the perspective of 664 00:28:11,210 --> 00:28:09,360 individual protein sequences and I think 665 00:28:12,620 --> 00:28:11,220 this will be a really nice way of us to 666 00:28:15,050 --> 00:28:12,630 try to bridge these two biological 667 00:28:16,970 --> 00:28:15,060 records that we have on the planet we 668 00:28:18,560 --> 00:28:16,980 have these material records such as the 669 00:28:21,260 --> 00:28:18,570 distribution of isotopes through time 670 00:28:23,030 --> 00:28:21,270 and we also have this molecular biology 671 00:28:24,290 --> 00:28:23,040 record and I think these isotopes might 672 00:28:28,250 --> 00:28:24,300 be a nice way to connect these two 673 00:28:30,050 --> 00:28:28,260 pieces of information together and the 674 00:28:31,760 --> 00:28:30,060 next place we're going with this in 675 00:28:34,130 --> 00:28:31,770 addition to that is to try to understand 676 00:28:36,470 --> 00:28:34,140 enzyme mechanism from the from these 677 00:28:38,090 --> 00:28:36,480 stable isotope fractionation x' this 678 00:28:42,080 --> 00:28:38,100 enzyme has a pretty cool proposed 679 00:28:43,820 --> 00:28:42,090 intermediate this is this is one of 680 00:28:45,200 --> 00:28:43,830 those intermediates in the proposed 681 00:28:49,370 --> 00:28:45,210 catalytic cycle and what you can see 682 00:28:51,260 --> 00:28:49,380 here is a fa d up here those four iron 683 00:28:53,510 --> 00:28:51,270 four sulfur clusters would be kind of 684 00:28:57,080 --> 00:28:53,520 off the stage floating up here electrons 685 00:28:58,970 --> 00:28:57,090 come down into the fa D and ApS is 686 00:29:02,030 --> 00:28:58,980 reduced leaving a covalently linked 687 00:29:03,560 --> 00:29:02,040 sulfite addict here so this is a pretty 688 00:29:04,480 --> 00:29:03,570 interesting proposed mechanism that 689 00:29:07,670 --> 00:29:04,490 hasn't received 690 00:29:09,650 --> 00:29:07,680 sufficient testing I don't think but one 691 00:29:12,620 --> 00:29:09,660 thing we're trying to do is use this 692 00:29:15,050 --> 00:29:12,630 proposed I this measured isotope value 693 00:29:17,240 --> 00:29:15,060 to test this proposed mechanism and I 694 00:29:21,470 --> 00:29:17,250 think that might be possible because the 695 00:29:23,060 --> 00:29:21,480 these ki es are kind of reports of the 696 00:29:24,440 --> 00:29:23,070 bond vibrations that can be expected in 697 00:29:25,430 --> 00:29:24,450 this type of environment so we might be 698 00:29:30,710 --> 00:29:25,440 able to approach that from a 699 00:29:32,210 --> 00:29:30,720 computational perspective in the 700 00:29:34,310 --> 00:29:32,220 beginning of the talk I said that this 701 00:29:38,210 --> 00:29:34,320 type of approach even though I'm going 702 00:29:42,230 --> 00:29:38,220 to talk about sulfate reduction it's 703 00:29:45,170 --> 00:29:42,240 it's very applicable to other microbial 704 00:29:48,260 --> 00:29:45,180 metabolisms we could look at some of 705 00:29:49,850 --> 00:29:48,270 Khmer Hasan's enzymes with a similar 706 00:29:51,650 --> 00:29:49,860 approach to look at the isotope 707 00:29:53,900 --> 00:29:51,660 fractionation of carboxylation or 708 00:29:59,150 --> 00:29:53,910 decarboxylation reactions we could also 709 00:30:00,860 --> 00:29:59,160 find interesting knowledge from testing 710 00:30:02,450 --> 00:30:00,870 other metabolisms like these apparently 711 00:30:04,340 --> 00:30:02,460 reverse metabolisms that I've put on 712 00:30:06,230 --> 00:30:04,350 here where one organism is putting 713 00:30:08,330 --> 00:30:06,240 electrons onto co2 to make methane and 714 00:30:11,990 --> 00:30:08,340 another one is taking electrons off off 715 00:30:13,730 --> 00:30:12,000 of methane to make co2 so I hope that 716 00:30:16,070 --> 00:30:13,740 this this way of determining enzyme 717 00:30:17,750 --> 00:30:16,080 specific apparent kinetic isotope 718 00:30:20,620 --> 00:30:17,760 fractionation factors will help us 719 00:30:22,280 --> 00:30:20,630 understand a lot of metabolisms and how 720 00:30:26,000 --> 00:30:22,290 sequences evolved through time 721 00:30:28,100 --> 00:30:26,010 catalytically and then when I was 722 00:30:31,040 --> 00:30:28,110 watching George's talk it occurred to me 723 00:30:31,430 --> 00:30:31,050 you know really enforced that if we're 724 00:30:33,380 --> 00:30:31,440 careful 725 00:30:35,690 --> 00:30:33,390 we will also be able to look at kind of 726 00:30:39,200 --> 00:30:35,700 these proto metabolic networks and if we 727 00:30:41,270 --> 00:30:39,210 can start measuring catalytic or rates 728 00:30:43,640 --> 00:30:41,280 rates of these types of reactions by 729 00:30:45,590 --> 00:30:43,650 different catalysts we'll be able to 730 00:30:48,260 --> 00:30:45,600 start to use these types types of 731 00:30:50,770 --> 00:30:48,270 isotope measurements to understand in 732 00:30:55,610 --> 00:30:50,780 more detail these non-biological 733 00:30:56,990 --> 00:30:55,620 reaction networks and so that's that's 734 00:30:58,540 --> 00:30:57,000 where I'm gonna stop with my talk and 735 00:31:04,010 --> 00:30:58,550 I'm gonna open it up for questions there 736 00:31:07,730 --> 00:31:04,020 and I just want to mention that this 737 00:31:13,090 --> 00:31:07,740 last year me batula Kochhar Daniel C 738 00:31:16,160 --> 00:31:13,100 gray Vaz wing Chris Bush and Chris house 739 00:31:17,980 --> 00:31:16,170 we were we started a proposal to look at 740 00:31:19,480 --> 00:31:17,990 ancient thio ester 741 00:31:22,450 --> 00:31:19,490 chemistry from a number of different 742 00:31:26,370 --> 00:31:22,460 perspectives one of them involves 743 00:31:30,610 --> 00:31:26,380 looking at promiscuity of thio ester 744 00:31:34,810 --> 00:31:33,430 how are you ki Tomi gave a really really 745 00:31:36,430 --> 00:31:34,820 fascinating talk a couple of days ago 746 00:31:38,830 --> 00:31:36,440 when he was talking and he mentioned 747 00:31:46,090 --> 00:31:38,840 that some enzymes are able to use an 748 00:31:49,600 --> 00:31:46,100 acetyl 2 amino ethyle I got it 749 00:31:51,010 --> 00:31:49,610 instead of acetyl co a as a cofactor and 750 00:31:53,140 --> 00:31:51,020 I think that is pretty interesting from 751 00:31:55,270 --> 00:31:53,150 an evolutionary perspective but we're 752 00:31:57,669 --> 00:31:55,280 also interested in making carbon and 753 00:31:59,410 --> 00:31:57,679 sulfur isotope measurements of these 754 00:32:01,000 --> 00:31:59,420 enzymes and trying to find out 755 00:32:03,970 --> 00:32:01,010 in a similar vein as what I presented 756 00:32:06,640 --> 00:32:03,980 here today how these isotope values may 757 00:32:09,400 --> 00:32:06,650 change through time or not and we're 758 00:32:13,060 --> 00:32:09,410 also interested in in trying to 759 00:32:14,860 --> 00:32:13,070 constrain how much thioester metabolism 760 00:32:19,090 --> 00:32:14,870 might be possible in a metabolic Network 761 00:32:21,190 --> 00:32:19,100 today or in the past so with that I'd 762 00:32:24,940 --> 00:32:21,200 like to thanks thank everybody for your 763 00:32:26,440 --> 00:32:24,950 attention and give a special extra 764 00:32:28,810 --> 00:32:26,450 acknowledgement to the funding that was 765 00:32:32,440 --> 00:32:28,820 supporting this process this progress 766 00:32:34,930 --> 00:32:32,450 and which was provided by NASA and also 767 00:32:41,639 --> 00:32:34,940 more recently from the WPI program here 768 00:32:59,169 --> 00:32:57,759 okay now the time for question so coming 769 00:33:01,810 --> 00:32:59,179 back to one of the themes of the meeting 770 00:33:03,610 --> 00:33:01,820 that there's always a lot of chemical 771 00:33:05,649 --> 00:33:03,620 stuff going on but maybe some of it 772 00:33:10,990 --> 00:33:05,659 matters and some of it is just kind of 773 00:33:12,159 --> 00:33:11,000 noise along the side the statement this 774 00:33:13,869 --> 00:33:12,169 is a question about whether this is a 775 00:33:16,090 --> 00:33:13,879 correct way to look at things your 776 00:33:19,180 --> 00:33:16,100 statement that sulfate is relatively 777 00:33:21,269 --> 00:33:19,190 unreactive species and so you have to do 778 00:33:23,769 --> 00:33:21,279 something unusual to it to activate it 779 00:33:26,499 --> 00:33:23,779 reminds me of what is the case also with 780 00:33:28,659 --> 00:33:26,509 nitrate which has tremendous capacity to 781 00:33:31,090 --> 00:33:28,669 reach oxidize but it's difficult to get 782 00:33:33,159 --> 00:33:31,100 at and it's a little bit like the 783 00:33:34,930 --> 00:33:33,169 Rubisco problem that co2 is not 784 00:33:36,399 --> 00:33:34,940 particularly reactive and it's not all 785 00:33:38,649 --> 00:33:36,409 that different from oxygens so 786 00:33:41,950 --> 00:33:38,659 activating it and distinguishing it 787 00:33:44,169 --> 00:33:41,960 discriminating it is hard all of the 788 00:33:46,720 --> 00:33:44,179 enzymes that do this seem to have big 789 00:33:49,720 --> 00:33:46,730 isotope signatures and the signatures 790 00:33:52,480 --> 00:33:49,730 seem to be related to how hard you can 791 00:33:56,379 --> 00:33:52,490 drive the reaction so we see things like 792 00:33:58,419 --> 00:33:56,389 a regular relation between isotope 793 00:34:00,249 --> 00:33:58,429 fractionation and the discriminating 794 00:34:03,129 --> 00:34:00,259 capacity and Rubisco x' and things like 795 00:34:06,039 --> 00:34:03,139 that but the great thing about these non 796 00:34:08,079 --> 00:34:06,049 reactive species is that vase they 797 00:34:10,569 --> 00:34:08,089 should be the chemical bottlenecks that 798 00:34:12,159 --> 00:34:10,579 wind up trapping free energy in one 799 00:34:14,440 --> 00:34:12,169 domain without a lot of reaction 800 00:34:16,270 --> 00:34:14,450 partners for it so that it can transport 801 00:34:19,180 --> 00:34:16,280 globally and become sort of a major 802 00:34:22,899 --> 00:34:19,190 carrier of disequilibrium should we 803 00:34:25,149 --> 00:34:22,909 expect that the major isotope signatures 804 00:34:27,579 --> 00:34:25,159 are the way they are because the major 805 00:34:29,859 --> 00:34:27,589 opportunities for biochemistry to take 806 00:34:31,510 --> 00:34:29,869 advantage of kinetic traps are these 807 00:34:33,099 --> 00:34:31,520 molecular like you know following 808 00:34:35,589 --> 00:34:33,109 avarice point are these molecular 809 00:34:37,000 --> 00:34:35,599 species that are hard to activate so the 810 00:34:38,319 --> 00:34:37,010 enzymes that can figure out how to do 811 00:34:43,270 --> 00:34:38,329 that are frequently going to wind up 812 00:34:45,730 --> 00:34:43,280 showing these kind of signatures I'm not 813 00:34:47,899 --> 00:34:45,740 sure I think that's I think that's a 814 00:34:52,460 --> 00:34:47,909 really great question 815 00:34:55,250 --> 00:34:52,470 I yeah I think there are they're 816 00:34:59,120 --> 00:34:55,260 probably examples of enzymes that are 817 00:35:00,950 --> 00:34:59,130 doing over overcoming well nitrogenase 818 00:35:02,089 --> 00:35:00,960 is one it's overcoming a huge kinetic 819 00:35:05,210 --> 00:35:02,099 barrier but it's isotope fractionation 820 00:35:07,819 --> 00:35:05,220 is really small and so it's it's hard 821 00:35:18,380 --> 00:35:07,829 for me right now to understand exactly 822 00:35:19,730 --> 00:35:18,390 how yeah what's general there yeah so 823 00:35:21,620 --> 00:35:19,740 that was actually the question I was 824 00:35:24,740 --> 00:35:21,630 just gonna ask you about nitrogenase and 825 00:35:27,920 --> 00:35:24,750 but I've got a second one he showed this 826 00:35:30,289 --> 00:35:27,930 huge spread of it wasn't your data but a 827 00:35:32,150 --> 00:35:30,299 huge spread of isotopic fractionation 828 00:35:34,730 --> 00:35:32,160 Xand people have tried to separate them 829 00:35:36,260 --> 00:35:34,740 out based on environment type and and so 830 00:35:39,380 --> 00:35:36,270 on and so forth but it has somebody I 831 00:35:41,329 --> 00:35:39,390 mean maybe this is obvious to people 832 00:35:43,490 --> 00:35:41,339 that do this work more but complete 833 00:35:45,109 --> 00:35:43,500 versus incomplete oxidizers whether or 834 00:35:48,230 --> 00:35:45,119 not they use cytochromes versus the 835 00:35:52,250 --> 00:35:48,240 sulfa vihren different major 836 00:35:53,839 --> 00:35:52,260 physiological variations and sulfate 837 00:35:56,240 --> 00:35:53,849 reduction that I believe are pretty well 838 00:35:59,180 --> 00:35:56,250 prescribed on to the evolution of like 839 00:36:00,920 --> 00:35:59,190 the the sulfate reducers themselves can 840 00:36:02,359 --> 00:36:00,930 you start backing out some of these 841 00:36:03,769 --> 00:36:02,369 patterns of fractionation based on 842 00:36:06,559 --> 00:36:03,779 cellular physiology and whether or not 843 00:36:09,200 --> 00:36:06,569 that tracks molecular in the evolution 844 00:36:10,880 --> 00:36:09,210 of these taxa I think that's a really 845 00:36:13,510 --> 00:36:10,890 great idea I'm not aware of anybody 846 00:36:16,640 --> 00:36:13,520 doing that in a comprehensive way that 847 00:36:19,010 --> 00:36:16,650 that Harrison and thawed paper from 1957 848 00:36:22,190 --> 00:36:19,020 has guided the field really strongly to 849 00:36:24,260 --> 00:36:22,200 try to really cell specific sulfate 850 00:36:26,539 --> 00:36:24,270 reduction rates to the observed 851 00:36:28,789 --> 00:36:26,549 fractionation and so people have gone 852 00:36:31,700 --> 00:36:28,799 out and measured many many types of SRB 853 00:36:36,170 --> 00:36:31,710 and only parameterised it against that 854 00:36:38,210 --> 00:36:36,180 rate but I think from what we know now 855 00:36:42,740 --> 00:36:38,220 especially from that model that Boz and 856 00:36:44,539 --> 00:36:42,750 etai constructed and then are and 857 00:36:46,279 --> 00:36:44,549 they're also recent permit permutation 858 00:36:47,990 --> 00:36:46,289 of that of being able to spread the data 859 00:36:50,210 --> 00:36:48,000 along an axis that is the amount of 860 00:36:52,609 --> 00:36:50,220 energy released and the reaction we 861 00:37:06,559 --> 00:36:52,619 should be able to do just that I think 862 00:37:10,109 --> 00:37:08,370 thank you for your talk 863 00:37:12,809 --> 00:37:10,119 he's very interesting so you have 864 00:37:15,120 --> 00:37:12,819 planned to apply the similar strategy to 865 00:37:18,329 --> 00:37:15,130 study the carbon isotope fractionation 866 00:37:21,390 --> 00:37:18,339 so in your case you is that a very pure 867 00:37:24,059 --> 00:37:21,400 system like like one and then or one 868 00:37:26,249 --> 00:37:24,069 organism and you what the molecule was 869 00:37:30,660 --> 00:37:26,259 the molecules that you hear try to 870 00:37:32,130 --> 00:37:30,670 target like carbon dioxide methane or or 871 00:37:37,319 --> 00:37:32,140 something like this it's a mineral 872 00:37:38,969 --> 00:37:37,329 record which data you a to write yes 873 00:37:41,269 --> 00:37:38,979 there's a couple yeah there's a lot of 874 00:37:43,169 --> 00:37:41,279 different it depends on the question 875 00:37:45,539 --> 00:37:43,179 wants to be addressed I think the 876 00:37:47,929 --> 00:37:45,549 autotrophic ones will be really 877 00:37:50,459 --> 00:37:47,939 interesting for example acetyl co a 878 00:37:53,849 --> 00:37:50,469 synthase synthetase would be a really 879 00:37:56,039 --> 00:37:53,859 interesting one to look at but in the in 880 00:37:59,039 --> 00:37:56,049 the cell even for non autotrophic 881 00:38:00,329 --> 00:37:59,049 pathways i think it we have to know what 882 00:38:02,669 --> 00:38:00,339 the rate limiting step 883 00:38:04,289 --> 00:38:02,679 is in a pathway and then we that helps 884 00:38:05,009 --> 00:38:04,299 us understand which one is the important 885 00:38:06,959 --> 00:38:05,019 one to look at 886 00:38:10,169 --> 00:38:06,969 and then for that question getting that 887 00:38:12,479 --> 00:38:10,179 enzyme specific number okay so the 888 00:38:15,449 --> 00:38:12,489 question will not be to explain the 889 00:38:32,120 --> 00:38:15,459 mineral record but to understand a 890 00:38:38,050 --> 00:38:36,440 right so if known so thank you very much 891 00:38:58,770 --> 00:38:38,060 [Applause]