SPEAKER 0
Okay, well, hello. And let me say, I'm happy to be here, if only virtually. And I really wish I was there with you today in Prague, one of my favorite cities. But nevertheless, I'm really happy to be able to tell you about our project on human mitochondrial manganese superoxide dismutase. And we have been working approximately seven years now on methods to detect directly coupled proton and electron transfers within this enzyme. So first off, I would like to describe to you the manganese side enzyme. And we've really over the years have always thought about it as the fountain of youth. So superoxide dismutase is are the first line of defense to protect organisms against toxic reactive oxygen species that damage our macromolecules of life and end up ruining our health. And this is really particularly important in the mitochondria, because the mitochondria are a hotspot for reactive oxygen species due to leakage of superoxide during the generation of ATP by the electron transport chain. So we're studying mitochondrial manganese, which is perhaps one of the more important superoxide dismutase. Now, overall, Sods have really fast reaction rates with a cake of over of ten to the minus nine, mole moles per moles per second. And this is because the enzyme includes electrostatic guidance to the active site. It's also able to transfer 40,000 protons per second. And I think by the end of the talk, I'll be able to tell you a little bit about why it's so fast. Sides always contain a metal ion. In the cytosol, it's a copper zinc enzyme, and in the mitochondria it is a manganese containing enzyme. And this enzyme is really important in health. It's really an obvious example is the hereditary forms of lateral sclerosis, which is also called Lou Gehrig's disease. These mutations cause a degenerative muscular neuromuscular disorder. So as we age, the levels of our antioxidant enzymes superoxide dismutase, as well as the downstream enzyme catalase go down, and we really just start to look and feel older. And that's why we call it the fountain of youth. Because if you just had more superoxide dismutase, you would live longer and healthier. So let's talk about superoxide dismutase. How is it that it has this diffusion limited? That the rates exceed a diffusion limited threshold. And what is it that contributes to its catalytic efficiency? So here we have the the the manganese side tetra colored in four colors with the four manganese active sites displayed. So this enzyme is really made up of a dimer of dimers, with right here being what we call two of the dimeric interfaces, with this being one dimer and this being another dimer. And here is what we call the tetra Maric interface. Now for this talk. It's the dimeric interface that's interesting to us. On the right here we have an electrostatic surface which shows the really positively charged active site, which makes sense if it's going to attract a negative superoxide anion. And here we have what I call the ring of fire, which are these negatively charged surfaces that actually repel superoxide towards the active active site funnel. Now if we take a cross-section of the active site funnel, it looks like this. And so we have some positively charged residues, a lysine here in arginine here that contribute to this positively charged active site funnel. And what really makes it positively charged is the manganese, which is either in a plus three or plus two state. Interestingly, we also have across the dimer interface this glutamine 162 I mean glutamic acid 162, which is actually negatively charged and would help guide repel the superoxide from this side over to the manganese. Now, when superoxide comes in, it has to pass through these two gateway residues, his 30 and tyrosine 34. So it would come in like this between these two gateway residues to the manganese active site. So it's not just diffusion, it's actually enhanced by the electrostatic guidance. And that helps it to be such a fast enzyme. I actually like to think of manganese side as a superoxide vacuum cleaner. And so here is the active site funnel again. And these these hair like things are the electrostatic vectors that show how superoxide is pulled towards the active site. And the active site would be down here. So these electrostatic vectors are actually going right towards that manganese ion. On the left here it's just some foreshadowing of the neutron results I'm going to show you today. Um, so let's talk about how manganese works. There's two half reactions. And so here the manganese in this resting state is in an oxidized state. And a plus three will be reacting with the superoxide which has an extra free radical. The manganese becomes reduced and oxygen is formed. In the second half reaction that reduced manganese becomes oxidized. There's a reaction with superoxide. And two protons come along and we form peroxide. So what are we asking? We're trying to figure out how this enzyme maintains a favorable electron transfer potential, meaning it's able to go from this redox cycling between these half reactions. And also protons move along with these electrons. And how does that work? So there's a coupled proton electron transfer that this enzyme uses to lower the activation energy of the reaction. And that's what we're really trying to understand is how do we get this coupled proton electron transfer. Now we know these protons pronation changes occur rapidly and really efficiently at physiological pH. And there seems to be a systematic proton relay that helps drive the reaction mechanism. And that's really what we want to see here. So to do this we're going to use a method that was new for me seven years ago called neutron crystallography. And so instead of using X-ray X-rays we're going to use neutrons. The reason for this is that carbon nitrogen oxygen and hydrogen really diffract neutrons equivalently. Now this is not true for X-rays. Hydrogens are very difficult to see with X-ray crystallography. And usually we really don't see them even at super high resolution. So we're going to use neutron crystallography to really see where the hydrogens are in the structure. So on the left here I have an X-ray map. And on the right I have a neutron map. In the X-ray map you can see there's a lot of white space. But in the neutron map it's really filled in on this tyrosine. We can see all the carbon hydrogens as well as this hydrogen off of the hydroxide or the oxygen. So also the water molecules we can see they're not just an X out in space there have we. We see their proton locations for the water molecules. And that'll be really important to understand superoxide dismutase. Here's an example from some of our maps where his 30 is a neutral in this state and has a proton in this state shown right here. And so that's actually a positively charged histidine. And this one is neutral. So we can see things like that in our maps. So to do this we're going to build a neutron crystallography pipeline for our project. And to do to start this out the first thing we had to do was replace all the hydrogens in the manganese side enzyme with deuterium. This can or cannot be easy to do. We have to change the expression system of for the enzyme. There is an added benefit that a fully iterated manganese sword is actually a slightly slower enzyme. So that might help our structural studies. So we went went for it and did this. The reason for it is in the neutron scattering cross-section, which is how each atom diffract neutrons. There is a coherent and incoherent part of the signal of the of the diffraction. The coherent piece is the signal where the signal is, and the incoherent piece is really, I think of it where the noise is. So if you compare hydrogen with deuterium, we see that the. Signal is actually three times higher for deuterium compared to. I'm sorry, it's three times higher than hydrogen comparing this direction, but the incoherent signal is 40 times higher for hydrogen versus deuterium. So what this means is the background is impossibly large. When you have hydrogen in your crystal. I shouldn't say impossibly, but it's very large. There's a 40 times decrease in the background noise when you replace all the deuterium in your crystal, in the protein, and in the solvent channels of the crystal, you get an incredible decrease in noise in your signal. So we were motivated to do that. And with the help of Kevin Weiss at Oak Ridge National Lab, we were able to express our manganese side in regular Beal 21 threes. We had to use that strain. A knockout strain did not work. And we grow the the bugs in deuterium minimal media which with a d glycerol as a source of carbons. The metals that we need in D tool and this is all published in this paper if you want to find out more. The second step here is to grow large crystals. Why? Because the neutron beam is relatively low flux. We need a bigger crystal. So we have more lattice repeats in the beam and we get more signal. So what we do is we set up very large drops. So here in this left side I have it's a clear sandwich box. So instead of a regular crystallization tray we set everything up in a sandwich box. The sandwich box holds one large reservoir and there are nine. There's a glass dish in here that's silicon ice. And there's nine drops of protein. Each protein drop is 100 microliters in size. And we have it at around 20 MiGs per mil. And you can see in this blow up that we were able to grow crystals in each of these drops. We were able to grow them the very first time we tried very large crystals. What we do is we just let them grow without touching in a vibration free environment. As we can get, we wait more than a month to ever look at them. The proteins per iterated. We have very simple crystallization conditions for this enzyme. Just a potassium phosphate solution which is easy to buy, iterated. And after the crystals grow, we use vapor diffusion with duty rated phosphate and D to zero to exchange all the hydrogens for a deuterium. So in the end, our crystals will have as much as possible every hydrogen replaced with deuterium. So on a very first try we got lovely crystals that are shown down below here, some of them quite large, 1.5mm cubed. We went to the beam line and shot the crystals, and it turned out the smallest one, which is only 0.25mm cubed, diffracted the best and neutrons the best. And we named her little bit Little Betty because she was a very nice crystal. Um, at the beam line, we use the Mandy beam line at Oak Ridge National Lab. This is one of the top state of the art beam lines in the world. It was run at that time by latent codes. So what we do is we put the crystal in the beam, and because we have this high symmetry space group, we are able to collect a full data set in only 6 to 8 frames of data. So this is a big advantage because each frame of data takes 24 hours. So we were able to collect a full data set in less than ten days, which sounds like a long time. But in neutron crystallography that's pretty good. Pretty fast neutrons are really precious, and so the Mandy beam line is really designed to collect complete data efficiently. It has a spherical array of detectors. It uses Laue diffraction so that more reflections are simultaneously refracting and it also separates those reflections in the time of flight to help reduce problems with overlap. So this is really a sweet beam line to use for neutron crystallography. So I'll propose to you that neutron crystallography is the ideal method to study metals in structural biology, for example metallo enzymes. Now why is this? The neutron beam is neutral to the metal. X ray beams aren't x ray. If you have a metallo enzyme, you put it in an x ray beam for data collection. You will reduce the metal during data collection no matter what, but the neutron beam is neutral and it doesn't affect the metal, so we can study a suitably oxidized manganese sad crystal structure for the first time ever. And as a bonus, there's really no radiation damage in neutron crystallography. So we can collect data at room temperature. So we set about to control the redox state of manganese side. Now to do this we mount the crystals in thick quartz capillary with thick quartz capillaries, not glass, but quartz because the glass contributes to a background in neutron crystallography. In the capillary we have two slugs of a permanganate solution, and in the middle here is our oxidized manganese crystal. You can see it's a really vivid red color because it's fully oxidized and has a high metal content. And we collected 2.2 angstrom resolution data off of this crystal that we named Ruby. We always name our data sets. We don't number them, we name them. So this is an oxidized manganese Saad data set. And it's high resolution because we only need two and a half angstroms resolution to really see deuterium positions. After we collected this data set. Then we replace the permanganate slugs with death ionized slugs. And so here's a picture of one of those crystals. Here's the crystal. Here's the die thing. And now this crystal turns clear so we know it's reduced in its manganese two. It happened to be the exact same crystal that we collected the Ruby data set with. So we have isomorphic data set. Ruby was converted to pebbles which we named that data set. And it's a 2.3 angstrom resolution data set. So after this data collection we then hand carry pebbles home to collect a corresponding x ray data set using our home source. And that's always necessary in neutron crystallography to have a paired x ray data set for refinement. So now we are going to look at the exciting part. Where are the protons? So let's take a look at the active site. Here is the active site manganese. And it's coupled with what we call the inner sphere amino acids. His 26, his 74, his 163 and aspartic acid 159. So these four amino acids are covalently ligand to the metal. We also also historically have always had this water molecule. We call it what one. In X-ray crystallography we can see that there's oxygen bound to the manganese. Then there's a second sphere of amino acids tyrosine 166 across the dimer interface. His 30 is hydrogen bonded. There we have a water molecule called what two. We have tyrosine 34 and glutamine 143. And remember the superoxide comes between his 30 and tyrosine 34. To get to the active site manganese. There's some additional amino acids, a glutamic acid and two tryptophan that are important for sealing off the active site, and hydrogen bonding with the other amino acids. So there's this hydrogen bond network that involves a water molecule and several titrated amino acids. The substrate and solvent all funnel through his 30 and tyrosine 34. The only ionized residues in this active site are his 30 and tyrosine 34. So it's always been thought that those two are important in the enzymatic mechanism. So what we're looking for here is how does it do this concerted proton electron transfer. So we have this electron transfer with an electron with its electron trumpet. And there's a concerted proton transfer that happens at the same time. So we're looking to see what proton transfers near the manganese can counteract the charge change that occurs when manganese three goes to manganese two. It must be something fast because we know it's a really fast enzyme. And so we're looking to see what the differences are when we change the redox potential between the two resting states of the enzyme. So now we're going to show you the neutron data and how manganese sun maintains its redox potential. And in these slides I always have the active site up here on the right with a circle around the part we're looking at and a reminder of the tetra coloring. Now remember these structures are without ligand. This is one of the most efficient enzymes. It's very fast. And this suggests that a proton must be primed for transfer. So let's look at what happens. Here is an oxidized oxidized. Crystal structure. Yeah. And we have manganese three here. This is our new neutron cross-section density map. I'm just going to call it a neutron map. And what we have is the two times f0 minus f c map at one sigma with some unbiased map density for the protons. So the protons were not a part of the structure during the phase calculation. So this is an unbiased map for the proton locations. And look at what one. This thing has plagued me my whole life. It's always been just an oxygen molecule. Finally, I can see in an oxidized state there is one proton on one and it's a hydroxide molecule. Glutamine 143 has two protons, as does tryptophan 123 and there's a nice hydrogen bond here to the glutamine. So the solvent molecule is a hydroxide in the oxidized state. Now let's look at the reduced maps. Look at this. What one now has a second proton. So there's our first proton transfer. What one becomes protonated. And where did it get it from. From glutamine 143. Glutamine 143 becomes a an anion. It has donated its proton to what one. That's why it's so fast. And look at tryptophan. 123 has become a very strong short hydrogen bond to help stabilize this neon. So the solvent molecule has become a water molecule. And we have a deprotonated glutamine 143 and stabilization by tryptophan 123 all of these observations were completely unexpected in that predicted for manganese superoxide dismutase, a deprotonated amide is really chemically unlikely, since they have such a high pKa, but we believe that being so close to this active site, metal has really changed the pKa of a glutamine. 143 and and so it becomes becomes deprotonated during the reaction cycle. We have done quantum mechanical calculations that really support these observations, and they're all in our publication from 2021. So how do the other amino acids contribute? Tyrosine 34 has always been thought to be important in the oxidized state. Is an anion. It's negatively charged. There's no proton in the reduced state. It's protonated. So it's protonated. During the manganese three to manganese two state, it becomes protonated. It loses its proton when it goes from manganese two back to manganese three. So perhaps this is one of the two protons that goes on to the peroxide product. Is is supported by this y 3004 F mutant that has been studied historically. That really stops activity only in this one direction going from manganese two to manganese three. So what else. Let's look at his 30. The N delta nitrogen of 30 becomes protonated when manganese side goes from a plus three state to a plus two state. We have a proton that's on here. Can see how what two has changed its orientation in the oxidized to reduce state. And there's this really unusual low barrier hydrogen bond that forms in the reduced state between tyrosine 166 and his 30. Now this is across the dimeric interface. It was completely. Unexpected. So we think there's this proton transfer that is occurring between, what, two going through his 30 to tyrosine 166 and then reversing and going the other direction towards what two in the reduced state. And perhaps this is a source of another proton to the peroxide product. So let me summarize what we've seen so far. Now remember we do not have substrate or product in our active site. These are just the resting state crystal structures of these three and manganese two in the oxidized manganese three state. What one is a hydroxide. Glutamine is in a hydrogen bond that with tryptophan 123 and one tyrosine 34 is an anion. It's deprotonated well and his 30 is in this interesting interaction with tyrosine. 166 upon reduction what one becomes a water molecule, glutamine 143 is deprotonated and becomes an anion. And there is a strong short strong hydrogen bond between tryptophan 123 and glutamine 143 that stabilizes this interaction. This proton electron transfer tyrosine 34 is protonated now. And there is this interesting hydrogen transfer between his 3166 and what two. And there's a neat I think it's neat low barrier hydrogen bond between his 30 and tyrosine 166. So in conclusion, I hope I've convinced you neutron crystallography provides valuable insight into the mechanism of this metallo enzyme oxidant reductase that no method to date has really approached. We've seen interesting things we never predicted in these crystal structures. We've directly detected concerted proton electron transfers in the active site in at several levels. We can see the proton relay, but within the water molecule structure and see that ionized amino acids near this active site metal have really altered PCAs. And I would put forward that maybe this is a common feature we will see in metallo enzymes is that the nearby surrounding active site residues have altered PCAs that contribute to the enzymatic mechanism. So in the future, we plan to use cryo cooled neutron crystallography not to save us from radiation damage, but purposefully to trap the superoxide substrate or the peroxide product in manganese crystals. Now, we're going to do this with wild type and also some strategic mutants that we know will help us to trap the substrate and product. So with that, I'll conclude my talk, and I really would like to throw out some acknowledgments to my colleagues at Oak Ridge National Lab and to the people who helped with the quantum mechanical calculations, but in particular to my graduate student, Jonas Admonish. He really has been the star of this project and really has done a large majority of the research I described to you today. So thank you very much, and I'd be really happy to take any questions you might have.