40th Annual Killian Award Lecture—JoAnne Stubbe
FULLER: So I want to welcome everybody to the annual Killian lecture, honoring the recipient of MIT's highest award for faculty achievement. I'm Mary Fuller, professor of literature and associate chair of the faculty. I'm standing in for Sam Allen. He wasn't able to be here today.
Before we get started, I have a couple of less glamorous announcements. First, please turn off your cellphones and anything that's likely to beep, if you would. And also, I've been asked to ask you to, if you would, move towards the front of the room and towards the middle of rows so that late arrivals will be able to be seated without too much back and forth, if you wouldn't mind.
It's a distinct pleasure and also an honor to introduce this year's winner of the James R. Killian, Jr. Faculty Achievement Award, JoAnne Stubbe, the Novartis Professor of Chemistry and Biology.
And our thanks are due to the Killian selection committee, chaired by Susan Silbey, for their work in proposing someone who's been both a profoundly dedicated member of the MIT community and one of the outstanding scientists of her generation, doing seminal work on the conversion of RNA to DNA.
Professor Stubbe was born in Champaign, Illinois. She received her Bachelor's in chemistry, from Penn, in 1968, and a PhD in organic chemistry from the University of California, Berkeley. She taught at Williams College, the Yale School of Medicine, and the University of Wisconsin before being lured to MIT in 1987.
And she's asked me to note that she was an assistant professor for 13 years at Williams. I think this also marks her 25th year at MIT. JoAnne Stubbe is a member of the National Academy of Sciences, the American Academy of Arts and Sciences, and the American Philosophical Society.
She's received many awards, of which I'll name only a few, the National Academy of Sciences Award in Chemical Sciences, 2008, the Benjamin Franklin Metal and Chemistry, 2010. Now I'm skipping over very long list to arrive in 2009, when she received the National Medal of Science, the highest domestic honor for a scientist, for work that provides compelling demonstrations of the power of chemical investigations to solve problems in biology.
In 2011, she also shared the Welch Award in Chemistry, with Christopher Walsh, for helping to explain how enzymes have evolved to carry out difficult and ingenious chemistry essential to life.
Now I'm just going to sort of stitch together some of the things that people have said in her nomination letters and also in the citations that's she's received for her work. More than any other scientist in the world today, JoAnne Stubbe has pioneered our understanding of the role of radicals in biology, those highly reactive molecules that catalyze chemical reactions at the cellular level.
A great deal of Professor Stubbe's research has focused on the ways that free radicals drive enzymatic processes, not only those that lead to aging and disease, by damaging DNA, but also the mechanisms for DNA repair and replication.
In particular, she has pioneered understanding of the enzyme ribonucleotide reductase, RNR, which catalyzes the conversion of nucleotides to deoxynucleotides in all organisms. Her research in developing a clear, unified picture of the forms, mechanisms, and functions of RNR has become textbook material.
Her nominating letter stressed not only the importance of her results but also the elegance, rigor, and creativity leading up to them. Her research draws on a vast arsenal of tools and techniques, from a range of disciplines, using them in innovative ways to solve new problems.
Professor Stubbe has been recognized for her fundamental research in biochemistry an enzymology. But her work also has powerful applications, in developing anti-viral and anti-tumor drugs, in particular an FDA approved drug that treats pancreatic cancer, and also environmentally friendly polymers.
This award also recognizes Professor Stubbe's dedication to teaching and her contributions to the curriculum at MIT. Soon after she arrived, Professor Stubbe was honored with the Teaching Award from the Graduate Student Council.
She has designed undergraduate classes in enzymology and biochemistry that have been enormously popular with MIT students and inspired imitators across the country. Most recently, she's collaborated in redesigning the introductory biology subject, 507.
She teaches students how to critically evaluate experimental results, internalizing the rigor that her own work exemplifies. Her impact on students, at all levels, has been profound.
One colleague wrote, she raises the bar and then gives students the tools to jump over it. Another colleague describes her as a mentor for junior faculty, who goes above and beyond in nurturing talent and providing opportunities for success.
Even while doing groundbreaking science and devoting herself to teaching and mentoring at all levels, Professor Stubbe has also found time, this year, to serve as a member of the Committee on the Undergraduate Program, where I had the pleasure of meeting her last week.
Professor Stubbe is both the quintessential MIT faculty member and, in the words of our late cherished colleague, Bob Silbey, one of the best biochemists in the world. Please join me in welcoming her to the podium.
Now, I'll just read the citation. This award is even to Professor JoAnne Stubbe in recognition of her dedication to teaching, the significance of her contribution to the biological chemistry curriculum at MIT, and the profound impact of her world on the field of enzymology.
STUBBE: Oh, wow, there's a lot of you out there. Thank you all for coming. I'd like to begin by thanking the selection committee and the faculty members who wrote supporting letters for my nomination. I know you have a lot of things to do with your time. And I'm grateful. And I'm really grateful to be the recipient of this award. I was pretty overwhelmed when Silbey informed me that I was the winner this past year.
And what I would like to do today-- I'd also like to thank MIT, as an institution, because I've had a fantastic place to be able to carry out my research, for the last 25 years, surrounded by really smart, creative people, with all kinds of facilities, where the sky is the limit in terms of what you can do. So thank you very much.
I'd like to begin by thanking the people who actually did all of the thinking and the experiments. And these have been my collaborators over the last 25, 35 years. And shown here are many of these collaborators.
But I want to point out a few of them from the Chemistry Department, the Nocera Group, Dan Nocera, Bob Griffin, and Cathy Drennan, who have played a role in the science that I will be talking to you about today.
It's wonderful to have collaborators that you can talk to, across disciplines, that are intensely interested in what you do at the molecular level. You don't find that very often anymore. So I'd like to thank them.
And of course, none of this would have been possible without the terrific students that we're able to attain at MIT. And this is my group shown in the top part of this slide. They're a bunch of really energetic, creative, hardworking people, who seemed to be able to put up with my insatiable desire for results. And there were continual requests for results, so thank you very much.
And down here, on the bottom part of the slide, are this Nocera Group that are working on the system I'm going to be talking about today. And over here is the Drennan Group that will be working on what I'm going to be talking about today.
And finally, I would like to thank my previous lab members, throughout all the years, starting at Wisconsin, where I had my first graduate student. That was back in the black and white era.
We were sitting on a 300 liter fermenter. Because back in those days, you could only isolate a microgram of protein as opposed to a gram of protein. There was not much molecular biology back in those days.
So "Radicals, Your Life is in Their Hands." This title was meant to be provocative. Many of you probably came because you didn't know what the title meant. And I hope that by the end of my talk you will understand what the title means.
So I want to start by defining the word "radicals." If you look it up in the dictionary, it has many definitions. But to me, what was immediately implied, when I thought about radicals, is my graduate school days, when I was at the University of California at Berkeley, in the late 1960s, where students were picketing against the Vietnam War.
And in fact, the establishment, the Reagan Administration, labeled radicals as protesters that were highly reactive, that one had difficulty controlling, and that wreaked havoc on everything they interacted with, namely turned over cars and smashed all the windows on Telegraph Ave.
But I would like to say, from my point of view, these radicals were really the inadvertent consequence of their immediate environment. And the immediate environment, in those protests, we're always the presence of a line a policeman that had guns raised and had gas masks on.
And so little did I think that these experiences, back in those days, would have foreshadowed the fact that I have spent 30 years of my life working on radicals in biology.
So what are radicals to a biologist? Radicals, again, are shown here. They're small molecules-- for today's lecture-- that have an unpaired electron. That's going to be indicated by a dot throughout my talk.
The electronic configuration of these molecules make them highly reactive. And molecules, like hydroxide radicals, react with whatever they encounter. For example, if they hit our genetic material, DNA, they can react and damage the DNA.
But what is the source of all these radicals that we find inside ourselves? The source is really the inadvertent consequences of our environment. So for example, when we're outside in the light, light can mediate radical damage to our genetic material.
We live in an atmosphere full of oxygen. And oxygen, when it gets inside the cell, can react with transition metals such as iron. They have the electronic configuration that allows them to readily react with oxygen or hydrogen peroxide, producing these reactive radicals, which then can mediate damage.
In addition, our normal metabolic pathways, when they get messed up a little bit, result in production of free radicals.
So almost all organisms have ways to combat these bad, free radicals. So nature has figured out how to evolve enzymes. These are the catalysts. Whoops. These are the catalysts of all the transformations that happen in our body.
This is the enzyme superoxide dismutase. The chemistry all happens at a little region within the protein called the active site. And these enzymes can actually mute the reactivity of these radicals.
In addition, many of you might have, on your kitchen table, bottles of vitamin C, vitamin A, vitamin E, antioxidants. You take these antioxidants to destroy these bad radicals.
But what happens if our methods of quenching the reactivity of the radicals fails? Then we have backups. So for example, if your DNA becomes damaged, we also have a battery of enzymes that figure out how to find the damage in our genetic material. They figure out how to cut it out. And they figure out how to repair it using deoxynucleotides, which you'll hear about in today's talk.
If these two processes fail-- and this happens on occasion-- over a long period of time, this damage can result in mutations, changes in our DNA, changes that are translated into proteins, and ultimately contribute to the aging and the disease process.
So radicals, in general, are thought about as being bad. Or they've been vilified, because they do destructive reactions. But today, what I want to do is talk to you about good radicals.
Good radicals are radicals that are generated using our catalyst enzymes in a very controlled fashion. And nature has figured out how to harness the reactivity of these radicals to do extremely difficult chemical transformations with exquisite specificity.
So I want to show you, in the next couple of slides, where you find radicals in biology. So again, most of you take vitamins. And if you've ever read the label on your vitamins, you'll see that half of the label is associated with organic molecules. These are vitamins that get transformed into co-factors that actually enhance the repertoire of the catalytic reactions that enzymes can catalyze.
And many of these vitamins, actually, in conjunction with the appropriate enzymes, can mediate radical dependent transformations. So here is vitamin B1, thiamine, vitamin B2, riboflavin, vitamin B6, pyridoxal, vitamin B12, adenosylcobalamin.
But what has been so surprising to me, from the last decade of research, in the age of genomics and bioinformatics, is another molecule that's not a vitamin, that's not on your vitamin bottle. And this molecule is called s-adenosylmethionine.
And undergraduates learn that this molecule is used to methylate, transfer a CH3 group to almost anything inside our cells. You can methylate a lipid or sugars or amino acids or nucleotides. But in fact, we now know, from detailed bioinformatics studies, that this molecule, itself, can generate radicals.
And if you look at the structure of this radical, it probably doesn't mean much to you. But it's the same radical that's generated by B12. So how are these radicals, in fact, generated? And this will become a take-home message from today's talk.
These radicals are generated by reaction with some kind of a metal center. And this particular group of enzymes, that use s-adenosylmethionine, or SAM, in this metal cluster to generate a radical, are called the radical SAM superfamily of enzymes.
And now, we know there are 2,800 members of this superfamily, all of which do radical dependent reactions. So if one looks at some of these reactions-- and most of them we haven't actually identified the chemical transformations they catalyze.
But let me just give you a flavor of some of the amazing transformations that these enzymes, with the use of radicals, can catalyze. So I just told you that light can damage your DNA. There is a radical SAM protein that uses free radicals to repair your DNA.
Many of you might be interested in the fact that there's a lot of methane gas produced by marine organisms in the ocean. People have postulated that the source was some kind of phosphonate derivative, that's a molecule with the phosphorous carbon bond.
And in the last two months, a paper came out, which showed that the methane gas is produced by a radical SAM protein, by cleavage of that phosphorous carbon bond.
If you look at another one of the vitamins on your bottle, biotin, it's made-- you have to stick an S, which is a sulfur, into this molecule, into unactivated carbon hydrogen bonds. This is very difficult chemistry. And in fact, it's a radical SAM protein that mediates that chemistry.
And finally, thiamine, this is vitamin B1 in the previous slide, does an amazing rearrangement reaction. And we don't understand the details of it, but it's also mediated by a radical SAM protein.
So radicals are everywhere in biological transformations. And the fact that there are now 3,000 of these things is going to keep people interested in unusual, important chemistry to the environment, busy for some time to come.
Now in addition to these small molecules that do radical-mediated transformations, there is another way you can produce radicals. And I think this really has also been under appreciated by the biological community.
So we've gone through an age of genomics, an age of proteomics. People are trying to figure out what all the proteins are doing. And now we realize, because of the amazing technology we have now, that proteins can increase their function and serve better regulatory roles by a process called post-translational modification.
That is after they're made, they get modified in some way with chemical entities. So many of you have heard of signaling by phosphorylation or histone code with methylation and acetylation. You have like glycosylation.
But I think many of you don't realize that you can also modify the amino acids on the enzymes or proteins, themselves, by oxidation. And so what you see now is that what people have found, over the last 20 years, is that your proteins can actually be oxidized.
So this is the amino acid tyrosine, which has been oxidized to tyrosyl radical. The dot being the radical. We found glycyl radicals. They're all part of the polypeptide backbone of the proteins-- and sulfur radicals and a tryptophan radical.
And today, we're really going to be focusing on tyrosyl radicals and sulfur radicals. And one of the questions that I want to focus on, at the end of my talk, is where do these radicals come from. How do you actually generate them?
So I hope I've convinced you, by these few slides, that radicals are everywhere in biology. So now the question is, how do we think about the kind of chemistry that they can catalyze when most of you, taking introductory course like 507, have probably not ever been exposed to radical-based reactions, even though I teach 507?
So what I want to do today is talk about a system that we've worked on for 30 years, ribonucleotide reductases. These are called RNRs. And these enzymes have really been a paradigm for thinking about radical-mediated transformations.
And what do these enzymes do? Well, they play a central role in nucleic acid metabolism by converting nucleoside diphosphates to deoxynucleoside diphosphates.
This is in the human system, which subsequent to phosphorylation-- so phosphate is these little purple balls-- provide the monomeric precursors required for DNA replication and repair. So you need four building blocks. And this one enzyme makes all of these building blocks.
And the key to the fidelity of DNA replication and DNA repair is the relative ratios of the deoxynucleotides pools and the absolute amounts. So if these relative ratios, of the orange to the green, get skewed, you end up having mutations in your DNA, which I've already alluded to, in the beginning, can result in mutations in the aging process.
So this one enzyme controls formation of all these deoxynucleotides and is regulated at every level imaginable. And so most people now, most biologists focusing on this system, are trying to understand the many, many layers of regulation that control fidelity. And I'm not going to talk more about that today.
But these enzymes also are central to making the building blocks of DNA. So if you wipe out this particular enzyme, you have no building blocks. And you can't make the DNA. And your cells die.
So this one enzyme is the only way, in all organisms, from methanogens, that grow on methane gas, to humans, that you can make deoxynucleotides. So we set out, many years ago, to try to understand the basis for this transformation.
And you might imagine, if you can inhibit this particular step, you might be able to design molecules that interfere with DNA replication, which then would produce, potentially, anticancer agents.
And I started out my career, when I was a faculty member, with potential to interact with students, at Yale Medical School, where I was interested in rational drug design.
So what is the actual transformation catalyzed by these systems? So this is a nucleotide. And I'm going to have a little cheat sheet over here. I know everybody gets mixed up with all the words of the dots and stuff, so you can keep coming back to the slide.
This is a nucleotide. And this molecule is a precursor to RNA. And the nucleotides is going to be converted by ribonucleotide reductases to a deoxynucleotide. These are the building blocks for DNA.
So what are you doing chemically? You're doing what appears to be, at least on paper, an extremely simple transformation. You're cleaving a carbon hydroxyl bond. And you're forming a carbon hydrogen bond. It looks simple. Nothing could be farther from the truth.
So what happens during this reaction? This is a reduction reaction. Two cystines in the active site-- these are amino acid cystines that look like that-- become oxidized, concomitant with the substrate reduction.
And to get multiple turnovers, to make a lot of deoxynucleotides, you have to have a way of reconverting the oxidized state back to the reduced state. And that's done enzymatically inside the cells.
But in addition to these two cystines that are required for the reduction process, there's a third cystine that has to be oxidized to a thiyl radical-- that I showed you several slides ago-- that plays an essential role in all ribonucleotide reduction reactions.
This was quite unexpected. And we were the first ones that demonstrated the existence of this kind of a species, that transiently exists, and it plays an essential role in this process, some time ago.
So what I'm going to do. I'll come back. And I'm going to talk about this cystine radical and its involvement in catalyzing this transformation. But first what I want to do is address the issue of this sulfur radical, which has a really short lifetime, where does it come from?
And it turns out that we now know, based on informatics and based on a huge amount of structural information, that there are three classes or ribonucleotide reductases. And the classification is based on the metal cofactors, which I'm going to introduce you to, that are involved in the oxidation of the amino acid cystine to this radical.
So what we know is that all three classes, I, II, and III-- and the one we're going to spend most time on, that's the one found in humans-- have the exact same structure. So they all have a common active site, where they have a 10-stranded barrel.
And there is the nucleotide that's going to be reduced. That's the nucleotide. Here are the two cystines, I showed you in the previous slide, that deliver the reducing equivalence. And they all have a finger-loop in the center of this barrel, with the cystine that needs to be oxidized to a sulfur radical to do any chemistry at all.
So I also have here a cloud. And this cloud is masking the radical initiator. So what I'm going to show you is, in these different classes, there is a different mechanism of oxidation in each class. And again, what I'm going to do is first introduce you to class II and III. And then I will spend a little bit of time talking about class I, which really has an unprecedented mechanism of long-range oxidation.
So if you look at class II, you've seen this before. This is B12, nature's most beautiful cofactor. And the function of this cofactor, with the appropriate enzyme, RNR, is to make a radical.
And here's the structure. We lift the cloud, and there we see the corrin ring, with a cobalt. That's the pink dot. And this ligand, which is the radical that does the oxidation of cystine to thiyl radical, sits within a few angstroms, about 3 angstrom, from the cystine that's going to be oxidized. So their close in three-dimensional space.
The class III enzymes are only found in organisms that are grown under strictly anaerobic conditions. They can't have any oxygen around. And here we have another molecule I've introduced you to earlier. This is the radical SAM superfamily of enzymes.
And again, it generates the same reactive species as B12. But in this case, the radical SAM enzyme generates a glycyl radical in the polypeptide backbone. Glycyl radicals are quite prevalent in biology.
And if we lift up the cloud over here, the same place where this cofactor is sitting is the glycyl radical. So this glycyl radical, now, is sitting within a few angstroms of the cystine that needs to be oxidized.
Well that's chemically reasonable. When you want to do a reaction, two things need to get together, and they need to be a few angstroms apart.
So what happens in the class I enzyme, which is the one found in humans? And in the class I enzyme, the observations are really quite striking. So first of all, if you lift off the cloud, what do you see?
You see two tyrosines. And these tyrosines are not redox active. They can't, by themselves, oxidize cystine to a sulfur radical. So where is the cofactor, the radical species that's going to catalyze our oxidation?
It turns out that this protein requires a second protein. And that second protein is called a subunit. So this protein houses these reactive intermediates. And this is a second protein-- have to come together to actually make your enzyme function.
So in this second protein, what do you find? You find this tyrosyl radical, I introduced you to at the very beginning of my talk, next to a diiron cluster. And I'm going to come back to the role of this diiron cluster in generating this radical at the very end of my talk.
What's amazing about this tyrosyl radical is the following. In solution, if you generate a tyrosyl radical chemically, it's lifetime is 10 to the minus 6 seconds. In this protein, in the choline protein, in the human protein, the lifetime of the radical is minutes to days.
And it's absolutely essential for catalysis. If you reduce this radical back down to the amino acid tyrosine, you can't make deoxynucleotides.
So what do we know? We know, from studies that have been done in the last six years or so, that, in addition to this well-studied class I enzyme, 1A enzyme found in humans, we've discovered two additional ribonucleotide reductases that sit in almost identical protein structures but have different metals.
And one of these-- and this was discovered by my lab in the last couple years-- we found a dimanganese tyrosyl radical cofactor that's going to mediate this oxidation. And in fact, we believe that pathogenic organisms are going to have a manganese cluster rather than an iron cluster.
In a study that was done six years ago, now, by Bollinger and Krebs, one of my former students, they discovered yet another metal cluster. There's no tyrosine. This is a phenylalanine that's hard to oxidize.
So you end up with a manganese-four and an iron-three. This is found in a human intracellular parasite. So what's amazing is that we've used three different metal motifs, that'll come back to at the very end of my talk, that are all this common structure, that are all going to be involved in oxidation of cystine to a cystine radical.
Now what's really striking about this is the chemistry is all happening over here. And you have a second subunit that has to come together, that has these radical initiators. And these guys are 35 angstroms apart.
Now, we just told you that most chemistry happens within a few angstroms. These guys are really separated. So what is the model for how that reaction occurs? So here is the subunit I showed you before, where all the chemistry happens, except now you're looking at all the protein spinach.
Here's the cystine and the substrate deeply buried in this protein. Here's the second subunit of this protein, which has these metal cofactors. And in this case, this is the class 1A enzyme, it has this tyrosyl radical.
So this structure is a docking model of these two proteins, which we now have quite good evidence for, based on my lab's collaboration with Cathy Drennan's lab and Marina Benatti's lab. And what's amazing is, again, this tyrosyl radical is removed 35 angstroms from this cystine.
So the question is, how do you do this oxidation over this long distance? And so the working hypothesis is shown on this slide. And this is the Nocera-Stubbe model for how this reaction works.
So here is the subunit, with the iron cluster. And I'm going to show you later on, its function is to generate this tyrosyl radical that's absolutely essential for catalysis. If you reduce it, you lose all catalytic activity.
Here is the subunit, over here, we've been talking about. Remember, I lifted the cloud, and I showed you that there were two tyrosines. Here's where the substrate binds, the nucleotide binds and needs to be converted into a deoxynucleotide.
So the proposal is, we know a lot about redox reaction in biology in things like the photosynthetic reaction center or the enzymes that convert nitrogen gas into ammonia or the respiratory chains.
We know electrons can move long distances, because they're tiny, by tunneling mechanisms, usually between two metal centers. But here, we have absolutely no mental centers. And the distance is longer than electrons can move to account for the rate of the nucleotide reduction process.
So the proposal is that this oxidation is going to occur by multiple steps, a hopping model, where we not only have to transfer an electron, but, because of the thermodynamics of these amino acids, we have to lose a proton.
So this process is called proton-coupled electron transfer. And it's a major focus of many people, now, interested in the energy problem when they're bio inspired. So let me show you then a cartoon. All this is meant to be is a cartoon of how this oxidation might happen.
So we need to transfer electron, for example, from this molecule to this molecule, and a proton. So the electron and the proton don't come from the same place. So we generated a new radical. And the proton was transferred.
Now we're going to generate another electron and the proton moves. Now, across this interface, which we don't know much about, because of the structural data we have, again, we're going to transfer an electron and a proton, an electron and a proton, an electron and a proton.
And here we are finally generating the sulfur radical, which actually mediates the chemistry. This is the same sulfur radical and a similar active site to the class II and class III systems.
And so here, we're now ready to convert a nucleotide into a deoxynucleotide. And at the end of this process, the whole thing goes back. And you reoxidize the tyrosine to a tyrosyl radical.
Now really, how stupid is this? I mean if you think about this, I mean you're talking about these amazingly reactive radicals. And there's so many places where you could make a mistake that you would think nature would never design anything like this.
I don't know, maybe one of you can answer this at the end of this lecture. But I don't have a clue.
So the question is, does this model have any basis in reality? So Nocera's lab and my lab have been studying this for eight years. And the answer is it does. The distance is extremely long. And very recently, using modern technology-- we never could have done these experiments, even five years ago-- we have evidence for transient tyrosyl radical intermediates, in addition to this radical, along the pathway.
So in the next two slides, I'm just going to show you sort of what the technology is that we've used to try to get evidence for this process. And for those of you can't remember what this weird pathway looks like, I've left it up there.
And so what we've been able to do now is to take advantage of modern technology, which allows you to replace any amino acid in your protein, this tyrosine or this one or this one or that one, with any other unnatural amino acid.
And so these are the tyrosine analogs we've been able to make and incorporate. And so how do you design your analog? Well, you're studying oxidation reduction. So one thing you want to perturb is the redox potential.
And so if you modify the number of fluorines in your molecule, you can change the redox potential over 300 millivolts. That's enough to turn off and on your enzyme.
If you want to study the proton coupling of the electron transfer, this is a tough problem, because electrons are tiny, protons are big. Electrons can tunnel over long distances. Protons can only move over short distances.
You can study what happens to this proton, again, by perturbing the proton transferability, which is called the PKA of your molecule. And so using these unnatural amino acids coupled to spectroscopy-- and again, this has been a collaboration with Bob Griffin's lab, with the Magnet lab, and also Marina Bennati in Germany.
We've been able to use high-field electron paramagnetic resonance spectroscopy, which allows us to look at all these radicals, and high-field ENDOR spectroscopy, which allows us to look at the radicals and then look at nuclei, the protons, interacting with each radical.
And that's what you have to look at to study proton-coupled electron transfer.
And I'm only going to show you one piece of data. Most people know that I love data. And my talks are usually all data. But I'm just going to show you an experiment we recently did, where we put this unnatural amino acid into this position.
So we put it in. We've replaced a hydrogen with an NH2 group. What that does is it makes this. This molecule is much easier to oxidize than tyrosine. And so, when you put the two proteins together, nothing happens. But when you add the substrate, you trigger off this electron transfer. This guy is going to get reduced to tyrosine. And this guy gets oxidized.
And so now it turns out that you can trap this radical. And this radical-- I think we were exceedingly lucky-- is kinetically competent to make the deoxynucleotides, the product of the reaction. And then we can use high-field EPR to look at the electronic properties of this radical.
And then we can use ENDOR, which allows us to look at the source of the protons and the interactions in the pathway. For example, this hydrogen and this hydrogen interact with that oxygen. And we've also unexpectedly observed a water during the electron transfer process.
So these kinds of experiments are really going to allow us to understand, I think, in chemical detail, the mechanisms of proton-coupled electron transfer. OK
So this is a very bizarre reaction. Are there any translational consequences? There are. I told you in the very beginning that there are three drugs used clinically in the treatment of cancers. And one of those drugs is hydroxyurea.
Many biologists in the audience might have used hydroxyurea. It's used in the treatment if hematalogical cancers. It's long been thought, to reduce this tyrosyl radical to an amino acid, if you reduce it, the enzyme's dead
But recent studies suggest, from my lab and Bollinger's lab, that, in reality, this molecule is probably interfering with this long range oxidation, over 35 angstroms.
There's a second molecule, tripine, in phase two clinical trials, which in the test tube destroys the tyrosyl radical and alters the iron cluster. And I think it inhibits ribonucleotide reductase inside cells. But it's early days, really, in terms of understanding the cytotoxic mechanism of this molecule.
So what I've shown you, then, is, I think, an amazing mechanism of oxidation. I'm now going to show you even more amazing chemistry. So we've been able to generate the sulfur radical. We've been talking about the sulfur radical. Let me put this back on.
We've been able to generate the sulfur radical. And what happens with the chemistry? So we started out, and our nucleotide, our nucleoside diphosphate, is being converted to deoxynucleoside diphosphate, the building blocks for DNA.
This is a reduction reaction. The two cystines in the active site get oxidized to a disulfide. And again, we have one, two, three cystines involved in this process. So for the first 15 years of my career, I spent a lot of time studying the detailed chemistry of this process.
And for today's lecture, I only want you to remember one thing. That the first step in this reaction is that this cystine radical, a reactive species, is sitting within a few angstroms of this hydrogen and can remove it to generate a nucleotide radical.
The rest of this chemistry is really complicated. I'm not going to tell you about it even though I love this chemistry. So what I'm going to focus on is this first step. And so the question you have to ask yourself is, how can you study something like this?
And so the approaches we took to studying the mechanism of nucleotide reduction is shown here. So for example, if your cleaving that bond, what you can do is replace it with a deuterium, a tritium, a heavier atom of hydrogen. It makes the rate slow down, and you can measure a difference of rates if this bond is cleaved. And that shows the bond was cleaved.
Alternatively-- and the days when we did this was the early 1980s-- you can change any amino acid-- this is the active site, there's the nucleotide-- to any other natural amino acid by site-directed mutagenesis.
For those of you young people in the audience, it used to take people something like five years to make a single mutant. That was your thesis. Now you can pull somebody in off the street, and, in two days, they can make 100 mutants. I mean that that tells you what the technology is like.
Anyhow, back in these days, we had to work pretty hard to figure this out. And what you could do is change an SH to an OH, for example. And if you did that, you would prevent this redox chemistry over here. This can't happen. And so you could potentially build up intermediates before that step actually happens.
So alternatively, what you can do, if you're a chemist, which I am, is that you can make analogs of the normal substrate. So all we've done in these analogs is change this OH into chloride or fluoride or azide or SH. So it's a pretty conservative substitution.
And what I'm going to show you is these guys became mechanism-based inhibitors, which we predicted based on our understanding of the chemistry, that I haven't talked about in detail.
So what is a mechanism-based inhibitor? And in the beginning part of my career, I was really focused on rational drug design. By understanding the mechanism, could I then use that mechanistic information to inactivate ribonucleotide reductase, which would prevent DNA biosynthesis?
So what I want to do is show you what a mechanism-based inhibitor is. So I've gone to a cartoon version. And what I want to do is talk about an enzyme called dolase. OK, dolase? Pineapple? OK.
So here's an enzyme. And this is the active site. And the substrate for this enzyme is a pineapple. So the pineapple binds in the active site.
And then the function of this enzyme is to cleave the stem from the fruit-- that's what the enzyme has evolved to do-- to generate stem plus fruit, which then dissociate off of the enzyme, allowing another pineapple to bind. And the process can repeat itself. So that's the enzyme.
How would you design a mechanism-based inhibitor? The first criteria, it must look like the normal substrate so it can bind in the active site. This molecule, the inhibitor itself is chemically unreactive. But using the normal catalytic mechanism, removal of the stem from the fruit, you're going to convert this molecule to a highly reactive species, because the chemistry is going to stray from the normal function of the enzyme.
So how would you design a mechanism-based inhibitor of dolase? So this is my rendition of a hand grenade. It looks like the normal substrate. It can bind in the active site. You remove the pin from the hand grenade, and all hell breaks loose.
I'm going to show you that, in fact, we can make mechanism-based inhibitors that look exactly like this hand grenade. So what happens, with these molecules, where are all I've done now is switch a hydroxyl group, over here, into a fluoride or an azide?
So we incubate them with the enzyme. And we spent a lot of time studying the detailed chemistry. So they look like the normal substrate, so they can bind in the active site. They're chemically very similar.
The normal mechanism, over here, is removal of a hydrogen atom from the 3-prime position. So the first thing that happens with both of these guys, which are chemically stable, is that you remove a hydrogen atom. Then after that, you loose fluoride. That's sort of analogous to losing water from that position.
With this guy, it's more complicated. Don't think about that. After you lose fluoride and azide, which turns into nitrogen gas and something else, what happens is now you cleave the base from the sugar. So you cleave that bond.
Furthermore, you lose an organic pyrophosphate. So look at what you've done by simply replacing a hydroxyl group with a fluoride. You cleave this bond, this bond, this bond, and this bond. The whole molecule is literally blown apart.
And what you're left with is part of the sugar. And this sugar species is what goes boom. This is a highly reactive molecule, activated for nucleophilic attack. And it kills the enzyme.
So the studies on these kinds of compounds told us a great deal about the chemical mechanism of ribonucleotide reductases as well as being potentially useful inhibitors. So let me show you one other example, which shows you the fine line between good and bad radicals.
So now what we've done is we've taken our protein, we've changed one atom out of 2,300 amino acids. We've changed this SH. We've changed a sulfur to an oxygen. And the sulfur and the oxygen, we've changed one of these guys that is involved in the redox chemistry.
Now what happens, the normal substrate becomes a mechanism-based inhibitor. You cleave this bond, this bond, this bond, this bond, generate the furanone, which inactivates the enzyme. And furthermore, you have a radical left at the end, which cleaves your polypeptide in two.
So you're walking a really fine line. We didn't do much to convert these radicals from something that we can control into something that was basically out of control. And so from a practical point of view, again, the studies that I just showed you, with the fluorinated compounds, were an inspiration to McCarthy's lab and Marion Dow Merrell, who designed Gemzar based on understanding of the chemistry of these reactions.
And gemcitabine is now a drug that's used clinically in the treatment of advanced pancreatic cancer and non-small cell lung carcinoma in combination with a wide range of other anticancer agents.
And it's a paradigm for mechanism-based inhibition. One equivalent of this compound wipes out the enzyme and kills it. And you get cleavage of that bond, cleavage of that bond, cleavage of that bond, cleavage of that bond, formation of this. And the enzyme dies.
There's a second drug, now, that is used clinically. This is a Genzyme compound called clofarabine. And we thought it was also going to be a mechanism-based inhibitor. We've been studying this for the last few years on the human enzyme.
It turns out not to be a mechanism-based inhibitor. But it's a slow tight-binding inhibitor. And what it does is it changes the quanternary structure of the human enzyme into a state where it becomes inactive. And we're in the process of actively investigating what's going on with this system in more detail.
So I've told you a little bit about the mechanism of nucleotide reduction. Now, in the last couple of slides, I want to come back to this question of where does this tyrosyl radical come from that is involved in oxidation of cystine to a cystine radical?
And what you see, I told you that this was on a second subunit from the subunit where all the chemistry happens. And what's really particularly interesting, from a chemical point of view, is we have two irons in a tyrosyl radical, two maganeses in a tyrosyl radical, a manganese and an iron.
And it immediately raises the issue of how do you control metalation inside the cell? How do you deliver the appropriate metal into the right enzyme given that, if you look at the environment of this metal cluster and this metal cluster, the ligands are exactly the same?
So I think a major unsolved problem in biological systems is trying to unravel the biosynthetic pathways by which metals are inserted into proteins. And I believe that you're walking a fine line. Remember, in the very beginning, I told you iron-2 can react with oxygen to produce bad radicals. But we need iron-2 in our protein to be able to oxidize tyrosine to a tyrosyl radical.
So my laboratory now has been focusing, in the last eight years or so, on the question of the chemistry. How does this chemistry happen in the test tube? And how does this chemistry happen inside the cell?
So in the test tube, we can take advantage of the fact-- and I think this is the reason why most people didn't really search very hard for biosynthetic pathways-- is that many metal clusters can self-assemble to form active species. They don't do it all that efficiently, but they can self-assemble.
But we can take advantage of that as chemists and study, using time resolved biophysical methods, the chemistry of oxidation of tyrosine to tyrosyl radical. So let me just put a tyrosyl radical over here.
And using these studies-- so this chemistry happens on a millisecond time scale. You can study the reaction using rapid freeze quench EPR, ENDOR, Moessbauer, and EXAFS spectroscopies. And I won't go through what these guys do.
But what it allows you to do is look at the details of what's going on with the metals during this reaction. And we've been able to detect, in the case of the class I enzyme, like in humans, the first iron-3, iron-4 species. Most of you are familiar with iron in the plus 2 and the plus 3 state.
And very recently-- it's still work ongoing in the lab-- we discovered, with a class Ib enzyme, which has two manganeses in it, the first manganese-3, iron-4 species that's involved in oxidation of this tyrosine to a tyrosyl radical.
So finally, let's come to the next to last slide. How does this happen in vivo? So in vivo, you have a protein. You make the protein. And there's no metal in it. So what do you need to be able to do?
In the end, we know, in the class Ia enzyme, we have two irons and a tyrosine that can oxidize to a tyrosyl radical. In the class Ib enzyme, you have two manganeses on that, again, can oxidize a tyrosine to a transl radical.
And how do you control this issue of mismetalation given that all the ligands around these metals are basically identical? And so what we now believe is that there's special equipment inside the cell. This has been very elusive for many people trying to understand this process of how do you deliver iron into the system and not manganese.
So if manganese goes into this protein, the enzyme doesn't function. It's dead. So you must have controlled metalation of your reactions. We know that, in addition to delivering the metal, we need a reducing equivalent. And we know that the oxidant, in this process, which eventually leads to oxidation of tyrosine to a tyrosyl radical, is oxygen gas.
However, with the manganese system, oxygen is not the oxidant. And what we discovered, several years ago now, is that this second protein, in the biosynthetic pathway, is going to specifically deliver the oxidant.
And what I'm going to show you is this oxidant is one of the bad radicals, superoxide. So what we've been able to do, in collaboration with the Rosenzweig lab, is get a structure of-- this is the protein with two manganeses in it.
The protein that is going to deliver the oxidant turns out to be a flavin protein. That's vitamin B2. And what you see, in the structure of the complex, so there's one protein. And the second protein, in the green, is this protein. And the gray is that protein.
What you see is a tunnel that goes from this co-factor. Flavin, in its reduced state, can react with oxygen, produce superoxide, which goes directly to the metal. It never gets out into solution, avoiding the problems with the bad radicals, namely superoxide. So nature has figured out how to control the reactivity of these radicals.
So to almost conclude, let me just summarize. I've told you that ribonucleotide reductase is really a playground for radical-mediated transformations. They have very unusual chemistry of nucleotide reduction involving formation of transient thiyl radicals and complex nucleotide radical intermediates.
I've showed you, in the class I enzyme, found in humans, this amazing oxidation, over 35 angstroms, through hopping tyrosine radicals.
I've also introduced you to the idea that you can have many different metal forms of this enzyme. And you really need to think about how to control metalation and oxygen delivery. Actually, 30 to 35% of all our enzymes have metals in them.
I've showed you, from a more translational point of view, that ribonucleotide reductase is the successful target or three drugs that are you used clinically. The cytotoxicity of these compounds is complex, involves many other proteins. But the most upstream protein is ribonucleotide reductases.
And finally, for those of you who think about evolution, I think this is a fascinating problem. Since Noller's work showing that RNA plays a key role in peptide synthesis in the ribosome, if you believe in an RNA world, how do you get to a DNA world?
The only enzyme that we've found, in all organisms, that convert nucleotides into deoxynucleotides is ribonucleotide reductase. You saw this weird radical-based mechanism. A chemist never would have done it that way. Why did we do this radical-based reaction?
And so finally, I don't want to leave you with the idea that ribonucleotide reductase are the only radical-based proteins. Tyrosyl radicals are found in many important places in biology. Many of you are now focused on the issue of the energy problem.
There is an amazing machine, in plants and in bacteria, that's capable of oxidizing water to oxygen gas requiring the presence of light. It's a manganese machine, maybe like the manganese ribonucleotide reductase. There are the four manganeses and a calcium.
What is this guy? This is a tyrosine. And transient tyrosyl radicals play an incredibly important role in this overall oxidation process. If you come over here to cytochrome oxidase, where is this protein found? It's at the end of a respiratory chain in all humans.
It does the opposite reaction. Except this one is thermodynamically favorable. It converts oxygen into water. And what does it do with all the energy that's released? It makes the energy currency of the cell, ATP.
So how does it do this? Here's a metal in the middle of this organic spinach, which is a heme. There's a copper, another metal. And here, you have sitting, right next to the center, a tyrosine attached to a histidine. And this is proposed to involve transient tyrosyl radicals.
And finally, if you go over here, to this little protein, this is COX, cyclooxygenase. All of you have had some kind of an experience with cyclooxygenase. It's the major target of non-steroidal anti-inflammatory agents and also of aspirin.
Here you have, again, a heme. And sitting right next to the heme is the tyrosine that mediates transient tyrosyl radical chemistry. So these radicals play a very important role in metabolism.
So radicals, hopefully you have some idea that they are highly reactive species that nature uses a lot. Your life is in their hands. I need not say more. If you don't have deoxynucleotides, you don't have life. So thank you.
Thank you. Sure.
FULLER: So I think we have a little bit of time for questions before the reception. If you'd like to ask one, please come to one of the microphones.
STUBBE: Oh, I recognize you. You work on ribonucleotide reductase.
AUDIENCE: Professor Stubbe, I was wondering, to make every molecule of dNTP, you have this oxidation of the cystines on an RNR molecule, which you have to reverse to make the enzyme functional again. This must be a very, very fast process, right?
STUBBE: Yeah, so the redox chemistry is very fast. The turnover number in the enzyme is quite slow, because the rate limiting step is all physical. They're all confirmationally gated.
AUDIENCE: Right, then how would DNA damage feed back onto this? Will it affect the rate of this proton-coupled electron transfer or will it just affect the number, the production of the enzyme?
STUBBE: What was the?
AUDIENCE: How will DNA damage--
STUBBE: So all DNA damage-- the relationship between ribonucleotide reductase and DNA damage is that, when you damage your DNA, you're changing the base of the sugar, the building blocks. And then you cut it out by some mechanism. You know there are many mechanisms.
And then what you need is just a deoxynucleotide to repair it. So it involves the DNA polymerases and the ligases. So it doesn't impinge. I'm not sure I understand your question. But it doesn't impinge directly on this electron transfer process at all. In other words, all this enzyme is doing is supplying the building blocks for DNA.
AUDIENCE: Thank you.
STUBBE: Hey, I recognize you, too. She's an RNA person.
FULLER: Can you come to the microphone?
AUDIENCE: If you express the class Ia enzyme in an organism that is normally from class Ic enzyme, do get manganese or iron found?
STUBBE: So the class Ic enzyme-- again, I didn't tell you the whole truth-- which has the manganese-4 and an iron-3, has only been studied in vitro. Because it's found in a human intracellular parasite, and you can't get enough of the material to actually look at the metalation.
In the case of the dimanganese and the diiron cluster, we pulled the enzymes out, under endogenous conditions, and show that we have either iron or manganese. But you know, I would dearly love to be able to do that experiment in the chlamydia system. But I think it's actually going to be technically impossible unless somebody invents some additional, really cool technology, which lets us look at things, maybe, inside the cell.
But the problem with ribonucleotide reductase is that they're there in very low concentrations. So it would be hard to use fluorescent spectroscopy or something like that to look inside the cell directly.
Uh oh, this isn't good. You always say this. When nobody asks any questions that means you didn't connect. I hope I connected a little bit, that you realize that there are really reactive radicals that can be controlled, and that you'll pay more attention to this in your future life.
FULLER: Thank you so much, Professor Stubbe. And everyone, there's going to be a reception downstairs, in the Barker room, room 105 in this building. So please join us.