00:00:30 Hello, welcome to this American Chemical Society satellite television seminar.

00:00:38 In this continuation of our annual series of conversations with Nobel laureates in chemistry,

00:00:43 we'll focus on the work of one of the 1997 prize winners, Paul Boyer,

00:00:48 who discovered the mode of action of ATP synthase,

00:00:52 an enzyme that has a key role in the energy transfer within our living cells.

00:00:57 We're pleased to have so many high school and college students, teachers, and professors join us today

00:01:02 to meet and talk with these Nobel laureates and other distinguished panelists.

00:01:06 I'm your moderator, Steve Lentz.

00:01:08 Our guest, Paul Boyer, who is Professor Emeritus in the Department of Chemistry and Biochemistry

00:01:14 at the University of California, Los Angeles.

00:01:16 Professor Boyer shared the 1997 Nobel Prize in Chemistry with John Walker of Great Britain

00:01:22 and Jens Skaal of Denmark.

00:01:25 To provide an additional perspective on ATP synthase research,

00:01:29 two of Dr. Boyer's colleagues have also joined us.

00:01:32 Dr. Roderick Capaldi is the Head of Biology

00:01:35 and a member of the Institute of Molecular Biology at the University of Oregon,

00:01:39 and Dr. Richard Cross is the Chairman of the Department in Biochemistry and Molecular Biology

00:01:44 at the State University of New York Health Sciences Center in Syracuse.

00:01:49 I'm here to host and introduce the program is the President of the American Chemical Society, Dr. Paul Walter.

00:01:56 Dr. Walter?

00:01:57 I'm proud to be a chemist and especially pleased to be able to host this program

00:02:02 highlighting some of the exciting and beneficial results of chemical research.

00:02:07 The evolution over the past 20 or so years of the understanding of ATP synthase functioning

00:02:14 provides a good example of several characteristics of science that I think are important.

00:02:20 First, science begins with the premise that truth exists and that it is knowable.

00:02:26 This belief in an absolute truth, discoverable by observation and experimentation,

00:02:31 is what makes science unique.

00:02:34 Second, although the problems scientists undertake to solve are at least partially determined by culture,

00:02:41 the end results of science are not culturally determined,

00:02:44 as are the results of studies in the humanities and social sciences.

00:02:49 Third, scientific research, industry, and concerns are global.

00:02:55 Chemistry is international.

00:02:58 And fourth, the results and applications of chemical research are frequently beneficial to people and society.

00:03:06 Chemistry has done more to reduce the death rate and improve the quality of life

00:03:11 than has any other profession in this century.

00:03:14 Dr. Boyer's model for how ATP synthase catalyzes the conversion between

00:03:20 ADP adenosine triphosphate and ADP adenosine diphosphate in living cells

00:03:27 was controversial when he first proposed it.

00:03:30 However, extensive research by his group and others in the international scientific community

00:03:35 demonstrated that it was correct.

00:03:38 Experimental work by John Walker and his group in Great Britain,

00:03:42 by Richard Cross and Roderick Capaldi, who are on the panel with us today,

00:03:46 and by Masatsuka Yoshida in Japan, among others,

00:03:50 confirmed Dr. Boyer's proposed mechanism.

00:03:53 This work crossed international and cultural boundaries

00:03:56 to result in a model that is now accepted as chemically correct,

00:04:00 thus illustrating the first three characteristics of science that I listed,

00:04:04 that scientific truth exists and crosses cultural and international boundaries.

00:04:09 The work that Roderick Capaldi will discuss later in this program

00:04:14 describes how this ATP research is being applied to treatment of human diseases,

00:04:20 illustrating my fourth point about the benefits of chemical research to individuals and society.

00:04:27 Other examples included in the background material

00:04:30 in the teacher's guides that you received for this program

00:04:33 discuss how researchers are continuing to build on the foundation of this ATP synthase research.

00:04:40 As the world's largest professional society for chemists,

00:04:43 the American Chemical Society helps scientists to keep building on the foundation of knowledge

00:04:49 started by those who have gone before them.

00:04:52 ACS fosters the advancement of chemistry,

00:04:55 promotes research in the chemical sciences,

00:04:58 and provides many vehicles for communication and education about chemistry.

00:05:03 We hope that today's program will help to increase your understanding about one aspect of chemistry,

00:05:09 raise your awareness of the need for all of us to have a basic literacy in science

00:05:14 to function as good citizens, and encourage you to learn more.

00:05:19 And now we'd like to hear from Drs. Boyer, Capaldi, and Cross.

00:05:24 First, Dr. Cross will provide a brief introduction before we hear from Dr. Boyer about his research.

00:05:31 What is it that makes ATP and its conversion to ADP special and important to study?

00:05:38 Well, ATP plays a central role in the flow of energy through living organisms,

00:05:44 and this flow begins with photosynthesis.

00:05:47 Photosynthetic cells have evolved the capacity to convert solar energy into chemical energy.

00:05:54 In this process, electrons are transferred from water to carbon dioxide

00:06:00 to give reduced fuels such as glucose and molecular oxygen.

00:06:05 Heterotrophic cells, which we're composed of, are incapable of this feat

00:06:09 and depend on the reduced fuels and oxygen produced by photosynthetic cells.

00:06:14 In tapping the energy that's stored in those cells, in those reduced fuels,

00:06:20 we convert the energy into ATP, and the fuels are returned to carbon dioxide and water.

00:06:30 ATP drives all the major work functions that our cells need to do to perform to stay alive,

00:06:37 and ATP drives all the mechanical work, most of transport work,

00:06:42 and either directly or indirectly, all of biosynthetic work.

00:06:47 In using the energy in the ATP, it's converted back to ADP and phosphate.

00:06:53 The structure of ATP is shown in the next diagram.

00:06:59 The ATP stands for adenosine triphosphate.

00:07:03 The adenosine part consists of an adenine ring on the top that's connected to a five-carbon sugar called ribose,

00:07:11 and the triphosphate part consists of a chain of phosphoryl groups

00:07:17 which are attached to one of the hydroxyl groups on the sugar.

00:07:22 This connection of the triphosphate chain to the sugar is an average phosphoester bond,

00:07:30 which is of normal energy.

00:07:32 However, the two bonds that connect the phosphoryl groups are special.

00:07:35 They're energy-rich phosphoric acid and hydride bonds.

00:07:40 When you add a water molecule across a bond between the second and third phosphoryl groups,

00:07:48 you produce adenosine diphosphate and inorganic phosphate,

00:07:53 plus energy that can do work to drive the work functions necessary to stay alive.

00:07:59 About 90% of the ATP that you drive from oxidizing reduced fuels

00:08:05 is produced in a process called oxidative phosphorylation.

00:08:09 And this shows a scheme for how that process occurs.

00:08:13 The oxidative part of oxidative phosphorylation is catalyzed by the respiratory chain,

00:08:19 which is shown on the right side of the slide.

00:08:22 The phosphorylation part is catalyzed by an ATP synthase,

00:08:27 which is shown in the central part of the slide.

00:08:30 Now, in coupling of the burning of fuels to ATP synthesis,

00:08:35 the respiratory chain first carries electrons from reduced fuels down to molecular oxygen,

00:08:41 and at several points along the way,

00:08:44 there's sufficient energy from the oxidation-reduction reactions

00:08:47 to drive protons across the membrane against a concentration and an electrical gradient.

00:08:54 This represents a storage of energy.

00:08:57 The protons cannot just dissolve back through the membrane, shown by the double line,

00:09:02 but can be carried, and the ATP synthase is able to carry the protons back through the membrane

00:09:10 and in the process use the energy that's available to make ATP.

00:09:16 The way in which this ATP synthase, or FOF1 as it's marked here, functions is a fascinating story,

00:09:24 and Dr. Boyer will now describe some of the experiments from his lab

00:09:29 which led to an understanding of the mechanism.

00:09:32 Thank you, Dr. Cross.

00:09:34 In the next diagram, you can see a scheme of the mitochondrion in the next slide,

00:09:41 which, as Dr. Cross indicated, there's a respiratory chain

00:09:45 where the oxygen you're now breathing is being used to make ATP,

00:09:50 doing it by making a proton gradient across an inner membrane of the mitochondria,

00:09:56 and when that proton flows across the membrane,

00:10:00 it catalyzes the formation of that terminal phosphoionhydride bond of ATP.

00:10:06 Now notice in the bottom part of this diagram that it splits out water.

00:10:11 If we reverse the process and hydrolyze the ATP,

00:10:15 it puts water into the phosphate, and phosphate has four oxygens.

00:10:20 Now a key part in our studies, a somewhat unusual approach,

00:10:24 made use of an isotope of oxygen, oxygen-18,

00:10:28 that we could determine then how extensively the incorporation of water oxygens

00:10:34 into phosphate and ATP occurred.

00:10:38 We found earlier in some of our studies a phenomenon we did not understand,

00:10:42 as shown in the next slide.

00:10:45 Here we're depicting the effect of an uncoupler,

00:10:49 a compound called 2,4-dinitrophenone,

00:10:52 which made it so the protons could no longer make ATP

00:10:56 and stop the oxidative phosphorylation.

00:10:59 And what was puzzling to us was that even though the oxidative phosphorylation

00:11:05 was stopped, as you can see by that sharp drop,

00:11:08 and the mitochondria achieved an ability to cleave ATP,

00:11:14 they continued to show a prominent exchange of phosphate oxygens with water oxygens.

00:11:20 And it was this resistance to this exchange that was puzzling.

00:11:25 And what then occurred to me at one time was that this could be explained,

00:11:30 as shown in the next diagram,

00:11:32 by considering simply a single catalytic site, as indicated here.

00:11:38 That if you bound phosphate and it could make ATP at the catalytic site,

00:11:44 if that reaction reversed, a water which could freely escape and re-add

00:11:50 would come and be present in the inorganic phosphate,

00:11:53 if the phosphate could go back on,

00:11:56 we would then have that exchange of phosphate-water-oxygen exchange

00:12:00 that became insensitive to the uncoupler.

00:12:03 This meant that our energy was still allowing that reversal reaction to go on,

00:12:08 the reversal of the formation of the ATP,

00:12:11 but was blocking the release of ATP.

00:12:14 So we thus then made the postulate, which was unusual at that time,

00:12:18 that a primary function of energy was to bring about the release of ATP

00:12:23 from the catalytic site.

00:12:25 Now we did a number of experiments to try to see if this was the case.

00:12:30 We found there was indeed a tightly bound ATP on the catalytic site,

00:12:34 as indicated in the next slide.

00:12:36 We turned to studies with chloroplasts,

00:12:38 because this ATP synthase is ubiquitous,

00:12:41 it's found throughout all of nature.

00:12:43 In our laboratory we used beef heart mitochondria,

00:12:46 spinach chloroplasts, Escherichia coli, E. coli,

00:12:50 a bacterial enzyme, as the principal sources for study.

00:12:54 And here in this particular experiment I'm illustrating

00:12:57 how we took reaction with chloroplasts,

00:13:00 shining light on them so that in that left syringe

00:13:03 they were making ATP, synthesizing ATP rapidly.

00:13:08 Then in the next syringe we put in another isotope,

00:13:12 P32, labeled inorganic phosphate.

00:13:15 Now if that is plunged,

00:13:17 that means we suddenly have P32 appearing in the reaction mix

00:13:22 and can see how rapidly it makes ATP.

00:13:25 We can use a quencher, a diluent solution,

00:13:27 and we actually put a filter in the way to trap the ATP.

00:13:31 And as shown in the next diagram,

00:13:33 we obtained data of this type.

00:13:36 We're now looking at a very short reaction time,

00:13:38 in the millisecond reaction time,

00:13:40 for photophosphorylation, making ATP.

00:13:44 And you can see that when we measure

00:13:46 the amount of enzyme-bound ATP,

00:13:50 it increases as we'd expect

00:13:52 as if it were an intermediate in the catalysis.

00:13:55 When we add an excess phosphate from another syringe

00:13:58 to dilute the radioactivity,

00:14:00 if this was indeed an intermediate,

00:14:02 it has to both form and disappear

00:14:05 in a catalytically competent manner.

00:14:07 As you can see in this diagram,

00:14:09 adding excess ATP to dilute the radioactivity,

00:14:12 the level dropped.

00:14:14 It was this type and other results

00:14:16 that gave us strong evidence

00:14:18 that a tightly-bound ATP at a catalytic site

00:14:22 was an intermediate and helped support the view

00:14:24 that energy was involved in the release of this ATP.

00:14:28 Now, as shown in the next slide,

00:14:30 we did other studies with the oxygen technique.

00:14:33 This one is a simple experiment

00:14:36 in which we were looking at the separated enzyme,

00:14:39 the F1 component that Dr. Cross mentioned,

00:14:42 which off the membrane will cleave the ATP.

00:14:46 And we show the rates of reaction

00:14:49 in three different panels.

00:14:51 The left panel, a very low substrate concentration

00:14:54 down in the range of micromolar.

00:14:56 The middle panel in a range from 1 to 20 micromolar.

00:14:59 And the top panel up to over 4,000 micromolar, 4 millimolar.

00:15:05 And the white line shows the effect on reaction velocity.

00:15:09 As we add more and more ATP,

00:15:11 it's cleaved more rapidly to reach a maximum

00:15:14 characteristic of many enzyme reactions.

00:15:17 But the unusual thing is

00:15:19 that as we lowered the ATP concentration,

00:15:22 the number of water oxygens

00:15:24 appearing in each phosphate increased markedly,

00:15:27 up to nearly four water oxygens incorporated in each ATP.

00:15:31 This meant that on that catalytic site,

00:15:34 the bound enzyme was reversing the cleavage of ATP

00:15:38 more than 400 times before it would release it,

00:15:42 and that the enzyme had catalytic sites,

00:15:46 multiple catalytic sites,

00:15:48 that participated in sequence

00:15:51 so that one catalytic site could not release an ATP

00:15:55 until ATP added in another catalytic site.

00:15:58 And that was a second feature of the binding change mechanism,

00:16:02 namely that we had release of a tightly bound ATP

00:16:07 and that multiple catalytic sites participated in sequence.

00:16:11 We also studied another aspect of the oxygen-18 techniques.

00:16:15 The next diagram shows what happened

00:16:18 when we measured the distribution

00:16:21 of what we called isotopomers that contained oxygen-18.

00:16:26 Up in the upper left in the green diagram

00:16:29 is the ATP that we prepared

00:16:32 containing highly labeled oxygen-18

00:16:35 so that most of the terminal phosphoro group

00:16:38 had three O-18s in it.

00:16:41 Now when we hydrolyzed this at the lower substrate concentration,

00:16:45 there was the exchange of the oxygens,

00:16:48 and we could then determine whether all the catalytic sites,

00:16:53 and by now we knew from the work in other laboratories

00:16:56 that there were three catalytic sites participating,

00:16:59 whether they all did the catalysis identically or not.

00:17:03 Shown here in the theory in yellow

00:17:06 is what we would have expected

00:17:09 if we had identical behavior of all three catalytic sites.

00:17:13 Shown in the right panel in the light blue

00:17:16 is what we'd expect if we had at least two pathways,

00:17:19 two different things, or if we had even more multiple pathways,

00:17:23 we'd expect other alternate distributions.

00:17:26 Shown in the panel of the light purple

00:17:29 to the left of the three is the experimental result.

00:17:33 And notice that the experimental result agreed closely

00:17:37 with that we expected if we had one catalytic pathway.

00:17:41 It was this type of data that told us

00:17:44 that all three catalytic sites were behaving identically.

00:17:48 And by now we knew that there were,

00:17:51 as shown in the next diagram, at least three beta subunits,

00:17:55 so-called large subunits, which had catalytic sites,

00:18:00 and that these then interchanged with catalysis

00:18:03 to proceed sequentially.

00:18:05 So they moved from a tight site, as shown in the lower right,

00:18:09 to an open or a loose site, as shown in the other diagrams.

00:18:13 And our postulate made at that time,

00:18:16 just at the beginning of the late 1980,

00:18:19 was that this occurred by rotational catalysis,

00:18:23 that it was the central part of that enzyme that rotated

00:18:26 that changed the beta subunits from tight to loose to open,

00:18:30 and this was a rotational catalysis,

00:18:33 again, something that had never been seen in enzymology,

00:18:36 but fit the data we had at this time.

00:18:39 And now, Dr. Walter, my other colleagues,

00:18:42 who also will take that and give instructions.

00:18:45 Let me interrupt just a moment, Dr. Boyer.

00:18:47 Thank you for the presentation.

00:18:49 We want to be able to get students involved in this,

00:18:52 so we're going to continue the discussion,

00:18:54 but we want to take just a moment in order to get you to call us

00:18:57 so that you can join in the discussion.

00:18:59 Please call at 888-802-6555.

00:19:05 For local callers in the 213 Los Angeles area,

00:19:08 that number is 466-3162.

00:19:12 When you call, an operator will answer

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00:19:18 Your site number is the order number listed on your invoice

00:19:22 or the packing slip.

00:19:24 You'll then be put on hold.

00:19:26 You will hear the program audio on your telephone,

00:19:29 and I will call on you by your name and location.

00:19:33 When you hear your location called,

00:19:35 speak into the telephone handset,

00:19:37 tell us your name, who your question is for,

00:19:40 and then we'll have several telephone lines standing by.

00:19:44 If you should get a busy signal, please hang up and try again.

00:19:48 And let me say to step away from your television monitor

00:19:53 if the phone is next to it, otherwise we get some feedback.

00:19:56 Remember, you must give us your valid site number

00:19:59 in order to ask a question,

00:20:01 and maybe I should repeat that phone number once more.

00:20:03 It's 888-802-6555.

00:20:07 So if we have some questions, I will hear those in my ear,

00:20:11 and why don't we do this.

00:20:14 If we get a question in a moment, we'll go to that.

00:20:17 Otherwise, shall we go on with Dr. Cross

00:20:20 speaking about his part of the program?

00:20:23 Oh, we do have a question?

00:20:26 Walter, go ahead with your question, please.

00:20:29 Okay.

00:20:31 This idea of rotational catalysis that you brought up,

00:20:34 is this unique in this particular example,

00:20:37 or are there other examples of rotational catalysis?

00:20:40 At the time that we postulated it, it was unique in enzymology,

00:20:44 with the exception that bacterial flagella,

00:20:47 a much larger apparatus, had been shown to rotate

00:20:50 and to be driven by proton-motive force.

00:20:53 So this was then a companion rotational machine,

00:20:56 but a much smaller rotational machine,

00:20:59 and one that was doing a chemical catalysis,

00:21:01 not a physical movement.

00:21:03 We also know at this time there's another closely related enzyme

00:21:07 in vacuoles that probably has this mechanism,

00:21:11 and some of the enzymes that are handling our DNA molecule

00:21:14 are circular and appear to have a motion of the DNA

00:21:18 driven by a cleavage of a triphosphate.

00:21:21 So nature uses the same thing more than once.

00:21:24 But for this particular kind of enzyme catalysis,

00:21:27 this was a first.

00:21:29 This was and is first, and at this stage,

00:21:32 it and the companion enzyme in the vacuole

00:21:35 are the only ones known.

00:21:37 Now, quite often when a scientist comes up with something

00:21:40 that's a first, it is not always immediately accepted

00:21:44 by the scientific community.

00:21:46 Was your analysis of these data accepted immediately?

00:21:51 I should have to say no it wasn't,

00:21:54 particularly the first suggestion that energy was required

00:21:58 for ATP release, because over the years

00:22:02 we developed the concept that energy had to go

00:22:06 to make the ATP molecule.

00:22:08 And the other thing, that there had to be intermediates.

00:22:11 Now we were doing away with intermediates

00:22:13 for a variety of reasons, and then energy was required

00:22:16 for the release.

00:22:18 It's interesting that when we submitted this for publication

00:22:21 in a leading journal in the field, it was declined.

00:22:25 So even a Nobel laureate was stuck in all of his publications.

00:22:29 Well, at that time I wasn't a Nobel laureate,

00:22:31 but I had been elected to the National Academy of Sciences,

00:22:34 and one of the privileges an Academy member has

00:22:37 is having papers published without review of this.

00:22:41 So the paper, A New Concept for Oxidative Phosphorylation

00:22:45 Based on a Molecular Explanation of the Isotope Oxygen Exchanges,

00:22:49 appeared in the Proceedings of the National Academy of Sciences

00:22:52 in 1973.

00:22:54 How did you get the idea initially that this was the answer

00:22:59 to the problem of ATP synthase?

00:23:02 How did you come up with this concept?

00:23:05 Well, we had accumulated over time this oxygen-18 data

00:23:11 and didn't understand what it meant,

00:23:14 this uncoupler insensitivity.

00:23:16 And frankly, Dr. Walter, I was listening to a seminar

00:23:21 on fine characteristics of magnetic resonance

00:23:24 of organic molecules that I didn't understand.

00:23:28 And so at that stage,

00:23:32 other things started whirling in my head,

00:23:35 the kind of things that you wake up thinking of in the morning.

00:23:38 And one of these was this unexplained data.

00:23:41 And it occurred to me, my gosh, we could explain that data

00:23:44 if the energy wasn't going in to make the ATP

00:23:47 that was going to release the molecule.

00:23:50 So at the end of that seminar,

00:23:52 I tried it out on some of my postdoctoral fellows.

00:23:55 And at first they didn't like it,

00:23:57 but it wasn't long before we realized

00:23:59 that this explained quite a bit of our data.

00:24:02 Well, I guess that shows that there's something useful

00:24:05 about going to boring lectures sometimes,

00:24:07 as long as we make the best of it.

00:24:09 Well, you have to have good luck,

00:24:11 but you have to have sort of a fertile field

00:24:13 to plant that luck in.

00:24:18 I'm fortunate that it came along this well.

00:24:21 Yes. Okay.

00:24:23 I believe we do have a call coming in from New Orleans.

00:24:27 If you can hear me right now,

00:24:30 would you please tell us who you are?

00:24:33 Are you hearing me?

00:24:36 Sir, I have a question on this flotation catalysis.

00:24:40 I think you talked about three subunits.

00:24:43 What is the state of the other subunits

00:24:46 when one subunit is involved in binding with an ATP molecule?

00:24:51 Is there any cooperativity in binding something like that?

00:24:56 You're asking about the function of the other subunits

00:24:59 on this complex molecule, if I understand.

00:25:02 Yes.

00:25:04 These are studies that are still being vigorously pursued,

00:25:08 and you'll see more of the structure of the molecule

00:25:11 here from Drs. Cross and Capaldi.

00:25:14 The answer is it's known a little bit

00:25:17 about what the big alpha subunits do.

00:25:19 They seem to have some regulatory functions.

00:25:22 The subunits that make up the FO part

00:25:24 are being intensively studied

00:25:26 to try to understand how the proton translocation

00:25:29 can make it so that this rotation occurs.

00:25:32 So what we know now is mostly about the catalytic portion

00:25:36 and something about the beta subunits,

00:25:38 and this then raises a whole host of new interesting questions

00:25:41 like the one you just posed.

00:25:44 Actually, why I ask this question

00:25:46 is because you talked about something tight, loose, and open.

00:25:49 So what I thought is something like if it is tight or open,

00:25:52 it's going to affect the other subunits.

00:25:54 That's what I thought.

00:25:56 Thank you for answering my question.

00:25:58 Thank you for calling in.

00:26:00 We'll keep the line open here if you think of another question.

00:26:04 Do we have any other calls?

00:26:07 Let me ask that first.

00:26:09 Okay, if not, shall we...

00:26:11 Dr. Walter, why don't you do a little introduction here

00:26:15 to Dr. Cross, and we'll continue.

00:26:17 Okay.

00:26:19 I'm very pleased to introduce our colleague Dr. Cross here

00:26:23 from State University of New York at Syracuse.

00:26:26 So, please.

00:26:28 Thank you.

00:26:30 Well, information that was made available

00:26:33 from the high-resolution structure of F1

00:26:36 that John Walker published in 1994

00:26:39 allowed us to design a critical test for subunit rotation.

00:26:45 The first scheme that we show here

00:26:48 shows F1 tilted backwards,

00:26:51 and the colors of purple and red and yellow

00:26:56 indicate the three catalytic beta subunits.

00:27:00 And the gamma subunit, which makes up the asymmetric core,

00:27:04 which you saw in one of Dr. Boyer's slides,

00:27:07 is shown in green coming out the bottom of the molecule.

00:27:11 So the idea that we wanted to test

00:27:14 whether this central asymmetric core rotates

00:27:18 relative to the three catalytic subunits.

00:27:21 And the high-resolution structure shown here

00:27:24 allowed us to design an experiment to test that

00:27:27 because it identified points of contact

00:27:30 between the gamma subunit and the beta subunits.

00:27:34 And you can see two molecules,

00:27:36 two atoms there highlighted in yellow.

00:27:39 The lower one is a naturally occurring cystine residue,

00:27:44 which occurs on the gamma subunit at position 87,

00:27:48 and the upper yellow mark

00:27:50 indicates residue 380 of the beta subunit.

00:27:54 So you can see these are very close together.

00:27:57 And the strategy that we used in testing for rotation

00:28:00 is shown on the next slide,

00:28:03 where we mutated the residue at 380.

00:28:08 If we could have the next figure.

00:28:12 This shows the cysts at position 87

00:28:16 on the gamma subunit on the upper left,

00:28:19 and the residue at 380 on beta,

00:28:22 the naturally occurring residue, is an aspartyl residue.

00:28:26 And you can see that they're close together.

00:28:28 This is predicted by the high-resolution structure.

00:28:31 Our strategy was to mutate that aspartic acid at 380

00:28:35 to a cystine, shown in the middle of the slide,

00:28:39 so that if we added an oxidant,

00:28:43 we could form a disulfide bond, shown at the bottom of the slide.

00:28:48 This S-S bond is a covalent tether,

00:28:51 which will permanently attach,

00:28:54 and it can be reversed by reduction,

00:28:57 but strongly binds the gamma subunit to the beta subunit.

00:29:01 And this allowed us to ask some interesting questions

00:29:04 about whether gamma rotation relative to the beta subunit

00:29:07 was required for catalysis.

00:29:10 The next slide shows the reaction that we ran

00:29:15 after we inserted this mutation at position 380 in the beta subunit.

00:29:21 If we added an oxidant, such as dithionitrobenzoate, DTNB,

00:29:27 we could rapidly form a disulfide bridge

00:29:32 between that central subunit, the gamma subunit,

00:29:35 and one of the three surrounding beta subunits.

00:29:39 Now, as predicted by Dr. Boyer's proposal,

00:29:42 that gamma has to rotate in the center.

00:29:45 Once we formed this disulfide bond, the enzyme was inactive.

00:29:49 But this did not prove rotation,

00:29:52 because it's possible that those two groups have to move closer

00:29:55 or further apart from each other during catalysis

00:29:58 without actually requiring a rotation.

00:30:01 So to answer that question, we did the following experiment.

00:30:05 The next slide shows that we formed a hybrid.

00:30:10 After we formed a cross-link

00:30:12 between the gamma subunit and one of the three beta subunits

00:30:16 at 10 o'clock in that figure on the left,

00:30:19 we then added... we dissociated the enzyme into subunits

00:30:24 and added the source of tagged beta subunit

00:30:28 and then reassembled the enzyme.

00:30:30 And now there's labeled beta subunit

00:30:33 in the two non-cross-link positions.

00:30:36 This allows us to distinguish the beta subunit

00:30:40 that is originally aligned

00:30:42 so that it can form the disulfide with gamma

00:30:45 from the other two.

00:30:47 And we then took this enzyme and put it back on the membrane.

00:30:51 This is the soluble F1 portion of the synthase.

00:30:54 And the next slide shows

00:30:56 that after we put it back on the membrane,

00:30:59 we reduced the disulfide bond,

00:31:01 and then we either turned the enzyme over,

00:31:04 allowed conditions for oxidative phosphorylation or not,

00:31:07 and then we re-added an oxidant to form the disulfide bond again.

00:31:12 And the question was, after turnover,

00:31:16 when we add the oxidant,

00:31:18 do we reform a disulfide bond to the original beta subunit,

00:31:21 which is not tagged?

00:31:23 That's shown on the left in the upper figure.

00:31:26 Or if gamma has rotated during catalysis

00:31:29 and stopped in different random positions

00:31:32 relative to the other two betas,

00:31:34 do we now cross-link the gamma to one of the tagged subunits?

00:31:39 Well, this is easy to determine

00:31:42 because the cross-link product runs on a gel

00:31:46 at a unique position with a molecular weight of 86 kilodaltons,

00:31:50 and the next slide shows the results.

00:31:53 Here we detected the tag

00:31:55 by using an antibody to the epitope

00:31:58 that we had put on the beta subunit

00:32:00 in the two non-bridge conditions.

00:32:02 And as you see in lane 2,

00:32:05 if during the brief time that the disulfide bond was reduced,

00:32:09 we just incubated and buffer,

00:32:11 there's very little tag in that 86 kilodalton band,

00:32:16 the second one down.

00:32:18 However, if we had conditions for ATP synthesis,

00:32:21 there's appreciable amount of tag

00:32:24 in that 86 kilodalton band in lane 1.

00:32:28 In fact, the amount that we see in this experiment

00:32:32 is close to what we'd expect

00:32:34 if the gamma subunit could react

00:32:36 with any one of the beta subunits

00:32:39 with an equal probability after turnover.

00:32:42 Next slide.

00:32:44 This provides compelling evidence

00:32:47 that gamma must reorient itself

00:32:50 relative to the three catalytic subunits during catalysis,

00:32:53 and you can see gamma in this figure

00:32:56 during one turnover turns 120 degrees

00:33:00 so that the pointy part of it

00:33:02 is pointing towards the open conformation

00:33:04 of the top subunit, beta subunit,

00:33:06 and gamma now moves around to the subunit at 4 o'clock

00:33:10 and causes the binding changes that Dr. Boyer talked about.

00:33:14 However, there are some things that our experiments didn't show.

00:33:18 We couldn't prove that gamma rotated continuously

00:33:22 in a 360-degree turn in one single direction.

00:33:26 Our results would have been consistent

00:33:29 with gamma just rotating 240 degrees

00:33:32 and then rotating back rather than going in one direction.

00:33:35 We thought that was unlikely,

00:33:37 but it was clearly important to demonstrate

00:33:40 that gamma rotates continuously.

00:33:42 And the next slide shows the design of an experiment

00:33:45 done in a Japanese lab headed by Masa Yoshida.

00:33:50 They took the F1 portion of the synthase

00:33:53 and attached it to a cover slip

00:33:56 through the beta subunit,

00:33:58 through little extensions that they engineered

00:34:01 into the beta subunit

00:34:03 so that the enzyme could not move.

00:34:06 Gamma, which is in the center of the molecule

00:34:09 and shown in yellow here,

00:34:11 during catalysis we'd expect that to spin.

00:34:14 What they did in order to observe that

00:34:17 was attach a long fluorescent-labeled actin filament,

00:34:21 which is shown in red in this slide.

00:34:25 And then they looked at the enzyme

00:34:30 attached to the membrane in a fluorescent microscope

00:34:34 and they photographed it as it was turning over

00:34:37 to see if they could detect the rotation

00:34:40 of that actin filament that they had attached to gamma.

00:34:44 And we now have a video that we'd like to show

00:34:48 which shows the result of that experiment.

00:34:52 Here you can see the fluorescent filament rotating

00:34:57 around the center of the molecule,

00:35:00 which you can't see because that's not fluorescent.

00:35:03 And this experiment was done by the Yoshida lab

00:35:06 in collaboration with some fluorescent microscopists.

00:35:10 And you can see it goes, it answers the question,

00:35:13 does it rotate in a single direction

00:35:16 under conditions for ATP hydrolysis?

00:35:19 And it goes through multiple 360-degree turns.

00:35:22 There's a saying that seeing is believing,

00:35:25 and this was a very dramatic experiment

00:35:27 that helped convince everybody

00:35:29 that that aspect of Paul Boyer's theory was right.

00:35:33 You can view this video yourself on the Internet,

00:35:37 and I think we can show you

00:35:40 the Internet access site for this at some point.

00:35:46 No, we don't have it quite yet.

00:35:48 We don't have it now.

00:35:50 Dr. Walter, do you have some questions for Dr. Cross?

00:35:53 I was just going to ask you,

00:35:55 what is it that is controlling the rate of rotation?

00:35:59 Well, you can see in this visual demonstration that Yoshida did,

00:36:04 the actin filament slows down the rotation.

00:36:07 In real, in normal functioning, this would be going faster,

00:36:12 but that long filament adds a viscous drag to the gamma turning,

00:36:17 so it's actually slower than the,

00:36:21 I think it was 100 millisecond time frames

00:36:24 that were taken in this video.

00:36:27 But normally what we think would happen

00:36:30 would be the gamma would rotate 120 degrees

00:36:33 and then pause while the substrates dissociated,

00:36:37 or the products dissociated,

00:36:40 or the substrates bound to the other two sites,

00:36:43 and then it would be ready to go again.

00:36:45 There would be a signal that the nucleotide sites are filled

00:36:48 and ready to go, and then the ethyl part in the membrane

00:36:51 would transport more protons and get another 120 degree movement.

00:36:56 Let me break in here just for a second to say

00:36:59 we want you to call us so that we can

00:37:02 at least get ready to take some more phone calls.

00:37:04 Please call at 888-802-6555.

00:37:09 In the 213 local area, it's 466-3162.

00:37:14 Tell the volunteer who answers the phone

00:37:17 your site number and your location,

00:37:19 and then we will put you on the air

00:37:21 with whatever your question is to one of the panelists here.

00:37:26 Dr. Cross.

00:37:28 I'd like to just comment a little bit more on the question that came.

00:37:32 You asked Dr. Cross because Dr. Yoshida's group

00:37:35 could actually calculate the energy required

00:37:40 to move that long actin molecule.

00:37:43 That's a long molecule.

00:37:45 As Dr. Yoshida said, if I'm the size of a gamma subunit,

00:37:49 that actin molecule is going out there a half a kilometer.

00:37:53 That's enough to make it visible so that you can see.

00:37:56 When you calculate the energy required to cleave that

00:37:59 compared to the energy needed to make an ATP for a third of a rotation,

00:38:04 the free energy you can get out from the hydrolysis,

00:38:07 they tend to be approximately equivalent,

00:38:09 meaning that it's an efficient machine,

00:38:12 that it's using the energy from the cleavage of ATP

00:38:15 very efficiently in doing that rotation.

00:38:19 Do we want to get Dr. Capaldi into the discussion right now

00:38:25 before we do his presentation?

00:38:28 Do you have a question you might ask?

00:38:30 Well, I'd like to ask Dr. Capaldi.

00:38:33 I think I'd like to hear his presentation first,

00:38:35 but then I definitely have some questions I'd like to ask him, yes.

00:38:38 I would like to ask Dr. Cross and Dr. Boyer another question right now.

00:38:43 What is it that drove you to work on this particular project?

00:38:50 How do you choose a research project like this?

00:38:53 Well, in my case, I was fortunate to have an opportunity

00:38:58 to do summer research at the University of Rochester

00:39:02 when I was an undergraduate student.

00:39:05 I studied with a professor called Isaac Feldman,

00:39:08 and we studied the mechanism of ATP cleavage

00:39:12 induced by divalent metal ions.

00:39:17 This was an inorganic chemistry study,

00:39:20 and we formed these complexes and studied the breakdown of ATP.

00:39:24 When I got to graduate school, I thought,

00:39:26 well, I've learned something about breaking down ATP.

00:39:29 I'd like to find out how it's made.

00:39:32 There happened to be one faculty member at the graduate school I went to

00:39:37 that was working on that project, and so I joined his lab.

00:39:41 I've been working on it ever since.

00:39:44 Dr. Boyer, what drove you into this field?

00:39:48 I was led into this field by the importance of the reaction.

00:39:54 Way back when I was in graduate school in 1943,

00:39:59 a couple of years earlier, Fritz Lippmann had first pointed out to the field

00:40:04 the importance of ATP as a currency of the cell.

00:40:09 It was then recognized that ATP had this central role.

00:40:13 Now that is over 50 years ago, you see.

00:40:16 At the time I was in graduate school,

00:40:20 a seminar was held, organized and held,

00:40:23 in the University of Wisconsin where I was at,

00:40:26 where I listened to some of the first evidence for oxidative phosphorylation,

00:40:32 that actually the amount of ATP that was made

00:40:36 made it necessary to have some way that was coupled to the oxidation reaction.

00:40:42 So this then posed the problem of oxidative phosphorylation.

00:40:47 In the 1950s and 60s, this became recognized

00:40:51 as one of the very prominent problems of oxidative phosphorylation.

00:40:56 Now I had the good sense not to study it then.

00:41:00 I actually worked mostly with other enzymes.

00:41:04 These protein molecules are so wonderful.

00:41:07 They can take these three-dimensional structures

00:41:10 to make a catalysis go that wouldn't otherwise go.

00:41:13 And I don't know an enzyme that I don't love, you see.

00:41:17 So I studied succinyl-CoA synthetase and alkaline phosphatase

00:41:21 and 3-phosphoglyceraldehyde dehydrogenase.

00:41:24 We did a lot of other work with enzymes.

00:41:27 But we always kept back on the burner, this oxidative phosphorylation.

00:41:33 Indeed, at the University of...

00:41:35 This is getting a longer answer, but at the University of Minnesota,

00:41:38 one of my fine research groups,

00:41:41 these fine postdoctoral fellows and grad students,

00:41:44 put in a remarkable effort trying to identify an intermediate in the reaction.

00:41:49 And we found a compound from P32 with a sensitivity

00:41:53 that was an intermediate between inorganic phosphate and ATP in mitochondria.

00:41:59 We thought we'd discovered the intermediate in oxidative phosphorylation.

00:42:04 We found out we were wrong,

00:42:06 that we'd discovered the intermediate in one of the substrate-level phosphorylations.

00:42:11 We were looking at part of the 10% of ATP is made, not the 90%.

00:42:16 In one sense, I say we were reaching for a gold but got a bronze,

00:42:21 because phosphohistidine, which we discovered then, was new in enzymology.

00:42:28 It was an important thing.

00:42:30 But it's like in research.

00:42:32 One of my other favorite sayings is that when you're doing research,

00:42:35 most of what you accomplish is the coal that you mine while you're looking for diamonds.

00:42:40 We accumulated quite a bit of coal.

00:42:43 But the magnitude of the importance of the problem kept me at it,

00:42:47 and this oxygen-18 technique that Mildred Cohn introduced it to

00:42:51 seemed to me like it should get us into the answer some way.

00:42:54 It finally did, but not until after decades later did we get to it.

00:43:00 We do have another call on the line from St. David's, Pennsylvania.

00:43:04 If you can hear me, go ahead with your question, please.

00:43:07 Good afternoon.

00:43:09 My name is Lynette Leung, and I'm coming from Eastern College.

00:43:13 I have several questions for Dr. Cross from a whole group of us over here.

00:43:20 Our first question is, what did you use to tag the beta-suppulant?

00:43:25 Yes, well, that's a good question.

00:43:27 We used different tags in different experiments.

00:43:30 Initially, we used radioactivity.

00:43:34 What we did was we grew E. coli in an incubation mixture

00:43:40 that contained a radioisotope of sulfur,

00:43:44 which got incorporated into the amino acids.

00:43:47 So those beta subunits were labeled.

00:43:50 We were then able to isolate those and use those as a source of beta

00:43:55 to replace the non-labeled betas in the two non-bridge positions.

00:44:01 That worked pretty well when we did studies of the soluble enzyme,

00:44:04 but when we put it back on the membrane

00:44:07 and we had all these other membrane proteins present,

00:44:10 it wasn't sensitive enough.

00:44:12 So what we did in later experiments was we put an epitope.

00:44:17 We engineered in an epitope into the N-terminus of the protein.

00:44:25 The N-terminus is exposed at the top of the molecule,

00:44:30 and it turns out that you can extend the N-terminus

00:44:33 with different sized pieces of protein,

00:44:36 and they have no effect on the activity of the enzyme.

00:44:39 So it didn't interfere with the normal functioning of the enzyme,

00:44:43 but it allowed us to put a tag which would react with a specific antibody.

00:44:48 We then used those betas to replace the unlabeled betas

00:44:54 in the two non-cross-link positions in the experiments that we did with membranes

00:44:58 because the antibody assay to detect their presence

00:45:04 was much more sensitive than the radioisotope assay.

00:45:09 So we used a variety of tags.

00:45:12 Is it Lynette? Did I hear correctly?

00:45:14 If you have another question, go ahead.

00:45:16 Okay. What antibodies did you use to identify these tags?

00:45:23 Yes, the tag that we attached is a tag that was commercially prepared by Eastman Kodak.

00:45:30 It's called the FLAG sequence.

00:45:34 The antibody that they produced, this is a kit.

00:45:38 You can buy the antibody, and they tell you the sequence that you have to insert in the protein,

00:45:46 and then you can use the antibody that they've raised,

00:45:50 and then isolate it, and they, of course, charge for this.

00:45:53 You can then use that antibody in an assay to detect its presence in the protein.

00:46:00 Do you have another question, Lynette, or does someone else there have a question?

00:46:04 Just one more question from our group.

00:46:07 We want to ask why the fluorescence chain rotated in only one direction.

00:46:14 We think that the enzyme, the gamma, will spin in one direction during hydrolysis,

00:46:24 and the Ushita experiment was done under conditions where the enzyme was running in reverse.

00:46:30 It wasn't attached to a membrane.

00:46:32 There was no proton current to drive it in the direction of ATP synthesis,

00:46:37 but it could run in reverse reaction because it's thermodynamically favored to hydrolyze ATP.

00:46:43 So they used ATP hydrolysis.

00:46:46 It would be very interested, and it hasn't yet been demonstrated,

00:46:50 but we would believe, according to Paul's proposals,

00:46:55 that during synthesis it would rotate in the opposite direction.

00:46:59 In that case, protons going down the gradient would drive the rotation in the opposite direction to make ATP.

00:47:09 When you go in the direction of hydrolyzing ATP, ATP would actually, if it were on the membrane,

00:47:14 would be pumping protons against an electrochemical gradient.

00:47:18 The synthase can run in either direction,

00:47:21 and probably whichever direction it's going will determine which way gamma spins.

00:47:27 Thank you very much.

00:47:29 Those are very good questions, and I'm glad students are getting involved in this.

00:47:36 Later in the program, I want to ask all of the panelists here to make some comments about students studying now.

00:47:44 How much does it take to get into a top-flight college?

00:47:47 We'll hear a few answers.

00:47:50 Right now, it's time to conclude the question period for this part of the program.

00:47:55 Hold your unanswered questions until the second half of the program.

00:47:59 We want to take a short moment to thank all the local site coordinators at each of the participating locations.

00:48:05 These programs would not succeed without you.

00:48:08 Whether you're at a university, a college, a school, or a company,

00:48:12 within an ACS local section or a student affiliate group,

00:48:16 we know that you have gone out of your way to make today's program a success for your group.

00:48:21 Please join me in applauding yourselves at the local site.

00:48:25 And now we're going to arrange for your group to receive this program.

00:48:29 It will be on videotape.

00:48:31 We'll be back for more discussions with our distinguished panel in about ten minutes from now.

00:48:36 Thank you.

00:49:01 ♪