 Welcome. Today I'm going to talk to you about a few of my favorite hemes and explain what a heme is in the second. This is a re-recording of a talk that I gave on March 13th, 2021 in Second Life at the Science Circle. I decided to re-record the talk because there were sound issues in the original recording and today I'm hosted on the chemistry world region of Second Life. So it's very close to the original venue. So I always include an abstract with my talks. You can read that at will. The abstract is also in the PDF of the talk which is available from the Science Circle website. I'd like to start with some acknowledgments. Of course, Science Circle, they've been supporting talks for many years now. Over a decade, many of which are recorded and posted on YouTube. I'd particularly like to thank the National Science Foundation for support for my own research as well as part of that support for the Science Circle. My research students, of course, are my good friends and collaborator, Dr. George Richteradu, who is my collaborator on the NSF. I'd also like to thank support from Southern Illinois University Edwardsville and everyone for coming and continuing to support the Science Circle in Second Life. So without further ado, today's talk. Well, I'm going to talk about what heme is in case you don't know. And heme is a part of many proteins that manage peroxide or transport oxygen or make nitric oxide or a variety of other useful functions in the body. And I'll go over and talk more about some specific proteins, hemoglobin, myoglobin, a little segue about how we know structure. And then I'll talk a little bit more about some selected proteins such as cytochrome P450 and neuronal nitric oxide synthase. So what is heme? Well, let me zoom in to a heme nitric oxide model. So this is a model that I uploaded into Second Life. It's based on x-ray crystallographic data that's available at the rcsb, here it is, rcsb.org from structure 4n8t. And coincidentally, that's a structure that one of my collaborator, George, and his student published. This is a heme molecule with a nitric oxide NO attached to it. So we just look at the heme part of it. You see it's an iron atom in orange. You see it's surrounded by a layer of four nitrogen atoms. And then there's the porine scaffolding of carbon atoms. I haven't drawn double bonds in, but essentially this molecule was aromatic, basically is planar. There is conjugation of double bonds all the way around this ring. And it satisfies the Huckel rule, 4n plus 2 electrons. Does that matter for today's talk? No, not really, but what it does mean is that there is extensive delocalization of electron density around the main part of the ring. Other features of their heme are that there are two residues that have carboxylate groups attached at one end. And these can be used for hydrogen bonding to proteins to help orient this planar unit in the molecule. On the other end, as you can see, there's only ethyl groups and methyl groups. So the other end is fairly nonpolar. So the iron likes to have six things attached. I've only shown five. In the actual protein, there would be another group that is linked to the protein in this vacant sixth site. So that's a heme. And specifically, it's a heme NO unit. My molecules that I've uploaded, if you take them, then you can get a note card from me that talks about where the structure came from and a few words about what the structure represented. So I've done that for every single structure for this talk. So moving on. Here are some examples of the active sites from some metalloproteins. I don't want to give the impression that heme is the only metal-containing active site in a protein. Here's an example of an iron sulfur cluster. This one is used for storing electrons for other sites to take advantage of. And there are iron sulfur clusters in molecules such as nitrogenase that takes N2 from the atmosphere and turns it into ammonia for nitrogen fixing. What I'm showing you today is going to focus more on the heme group. So let's see. This guy up here is a heme from a cytochrome P450. Here's a heme from a hemoglobin and a myoglobin. And here's a heme from a catalase. And the heme themselves, the flat iron porphyrin, the organic part of the heme is a porphyrin, is the same pretty much from structure to structure. You'll notice there's some details that are different, especially in terms of how the carboxylates are oriented. And a big detail is what is in the coordination site below the heme. I'm going to call that the proximal site. For the cytochrome P450 and things like neuronal nitric oxide synthase, you have a cysteine. And the cysteine is a protein. It's got a sulfur donor. And this cysteine unit would be part of the larger protein polymer. I'll show you examples of those later. Hemoglobin, myoglobin, and some peroxidases have a histidine unit. That's basically, it's got an amidazole ring. That's this five-membered ring with two nitrogen, not beside each other. That's in that last space. And then something like a catalase might have a tyrosine, which has a benzene ring with an OH. And here the OH is deprotonated. And the oxygen is directly attached to the iron. So these molecules are ubiquitous in biochemistry, but they're not in every single protein that's out there. So a couple of things. I've mentioned catalases, excuse me, catalases and peroxidases. These things catalyze the reactions of H2O2. H2O2 is made naturally in your body. You basically make it as a consequence of your metabolic activity. It is a strong oxidant, and so having it in your body in the wrong place at the wrong time can be a very big problem. It would contribute to oxidative stress in your metabolism, and that can lead to cell wall degradation or mutations or just generally bad stuff happening. So there's two ways of getting rid of this stuff. And one way is simply to use a catalase, and you've all got catalases inside of you, and catalase will take the two molecules of hydrogen peroxide and convert them into water and good old dioxygen. Dioxygen in the wrong place at the wrong time is not much better, but at least it can diffuse and perhaps get onto a hemoglobin and be transported somewhere else where it can be of use. The other thing that we can do with hydrogen peroxide is use a peroxidase. And a peroxidase actually takes advantage of the oxidizing power of hydrogen peroxide to make some other chemical transformation happen. Here, AH2 is some molecule. It's got two hydrogens on it, and those hydrogens end up becoming attached to the oxygens that came from H2O2, and then you're left with some molecule A. In other uses of this peroxidase enzyme, you could have A with a double bond O attached to it as well. So basically it's an enzyme that can oxidize some substrate which will be used in some other biochemical process a little bit later. So talking about catalases and peroxidases, though essentially what we have here is the active site that you would expect to see with a catalase and the active site you would expect to see with a peroxidase. Notice that the catalase has a tyrosyl unit attached to the iron. Also notice that I'm representing the four nitrogens of the heme group by the vertices on this square. So catalase has a tyrosyl unit, peroxidase has a histidine unit. That's very similar to hemoglobin and myoglobin. Hemoglobin also have histidine units down here below the plane of the heme in what we're calling the proximal site. Where the action happens at these enzymes though is in what's called a distal site. And both of these have histidines that are close to where something would bind to the iron atom, but not too close. They're close enough so that hydrogen bonding can help to orient whatever is attached to the iron when the iron is doing its business. There's also another hydrogen bonding, basically aspartic acid I think or an aspartate or an arginine in the case of peroxide. So these units up here with hydrogen bonding help to keep the substrates and H2O2 in their proper orientation for these enzymes to work. So catalase, catalase takes H2O2 turns it into water and dioxygen O2. How does that work? Well the first thing that has to happen is that the H2O2, the hydrogen peroxide has to bind to the iron. There it is bound to the iron. And the histidine and the aspartate or arginine hydrogen bond to both ends as you can see. There's a hydrogen bond there and a hydrogen bond there. They bond to both ends of the H2O2 unit. What happens next? Well the combined action of being bound to the iron and having all this other hydrogen bonding encourages a bond to form between the oxygen that's further away from the iron and this hydrogen. The other thing that happens is that the oxygen-oxygen bond breaks. So we end up with a water molecule because now both hydrogens are attached to that oxygen. And then this unusual iron double bond O is an iron plus 4. Looking at that it's got an iron plus 4 oxidation state. That's a very oxidizing form of iron. Usually iron 2 and iron 3 plus are the forms that you would encounter. Iron 4 plus is a very rare form. And that's not all. This structure is called compound 1. It has another oxidizing equivalent because we have removed another electron from the heme itself. So somewhere from the what we'll call the pi system these delocalized electrons around the ring of the heme there's one missing. The upshot is that this form of the molecule can remove two electrons from something else in a single reaction. And that's what happens next. So what happens is that once we get into this state another H2O2 comes in. Electrons are ripped off of it essentially. And we form an oxygen or electrons are ripped off of it. The hydrogens go away H plus and essentially protonate the oxygen that's attached to the iron. And so you're left with water and dioxygen. The little star represents where those oxygens end up. So the O2 all the oxygens in there came from that second H2O2. So this compound 1 is very powerful oxidant. It took a long time to isolate and be observable. But it's pretty well established now that this pathway or this intermediate in this pathway is a real thing. So what I can show you there we go. I'm going to show you some horseradish peroxidase fairly soon. Here we have a heme group that is in the compound 1 state. It seems to be getting mixed up with some other thing. Let me move that out of the way. Okay so here we have a heme group. It's in the compound 1 state. This is a single chain of a protein. So the blue starts the blue starts right here. There's one end of the blue and this spaghetti and spirals and sheep is one single polymer. It goes nitrogen, carbon, carbon, nitrogen, carbon, carbon, nitrogen, carbon, carbon with other things hanging off of those atoms provide functionality. So anyway it's one single chain. You can follow it in an unbroken line from start to finish. Along the way, I'll notice it looks broken. It looks broken here but it's not because I've actually put the histidines in explicitly. So the histidine, you can tell it's histidine because it's got a five-membered ring. This histidine is part of the chain up here. This particular histidine that I'm showing you right now is one of those histidines that helps to orient the H202 in the active site pocket. Here we have the iron oxo unit of the compound one and in the bottom you can actually see another histidine. I didn't draw a bond there but there is one and then again you can see the gap in the spiral where the amino acids are not shown because I've explicitly put the atoms in for this particular part. There's calcium in here and none of these proteins I've shown the water. There's water everywhere in all of these proteins. In fact, the calciums are bonded to six waters in the structure. There's also acetate coming from the conditions used to crystallize this. This is an x-ray crystal structure so it's pretty well established that this compound one state is a thing. It's pretty well established in the literature. Horseradish peroxidase is also produced locally. I am near St. Louis and Milliport Sigma is a major company here and the area grows horseradish so horseradish peroxidase can be isolated from local materials. Next slide. I've talked about hydrogen bonding. I just want to talk about that a little bit more. Hydrogen bonding is basically an intermolecular force. If you think of things like normal CH bonds or carbon-carbon bonds, they arise from sharing of electrons between two atoms. A hydrogen bond is basically an electrostatic type of bond. If you look at an oxygen atom, it has a very strong attraction for electrons and hydrogen has a somewhat weaker attraction for electrons. So when the two are attached to each other, electrons tend to spend most of their time around the oxygen atoms and they tend to neglect spending time around the hydrogen. That ends up giving the oxygen a net permanent negative charge and the hydrogen a net permanent positive charge. Well, these dipoles in these molecules end up attracting each other. In fact, a water molecule has two lone pairs and each of those represents two concentrations of negative charge. It's got two OH bonds. Each hydrogen represents positive charge. So a water molecule can hydrogen bond in all four directions, essentially along its tetrahedral shape. So what is the strength of hydrogen bonding? Well, it's like one tenth to one twentieth of something like a carbon-carbon bond, I guess. So each hydrogen bond is not particularly strong, and that's a good thing because if they were really, really strong, the bonding would be irreversible. Hydrogen bonding is a feature of DNA. If you think of the rungs in DNA, each rung, the sides are held together by either three hydrogen bonds or two hydrogen bonds. Each rung isn't held together by extremely strong forces, but when you have a hundred or a thousand of these rungs, they add up and keep the DNA quite stable. Just unstable enough to be unzipped and allowed to react when appropriate. Here's my slide on the horseradish peroxidase. Basically, here's a view of the active site that I just showed you. It's structure 1HCH from the RCSB PDB. Basically, it's from that particular structure. I won't go into the chemistry too much, but essentially horseradish peroxidase can take substrates and oxidize them. Organic substrates can be oxidized. One favorite one that people have is camphor, and then it can be turned into a ketone. So horseradish peroxidase, because it's so available, it finds its way into commercial products, finds its way into a lot of different studies. So it's one of my favorite heme containing enzymes. Another favorite because I enjoy being alive is heme globin. Here are, here is some content from the PDB. Basically, PDB 101 has some really nice information on heme globin. Here you can see the heme group in one of the four subunits of a heme globin. You can see the histidine bound to the iron. Notice how the iron is not quite in the plane, and when an oxygen molecule, and this is the regular O2, it's not hydrogen peroxide, binds. Then the iron pops back into the ring. Maybe it pops out on the other side a little bit. So heme globin is a tetramer. It's basically got, well actually it's got an alpha subunit and a beta subunit. They're almost the same. Each contains a heme, and then a pair of those subunits dimerizes with another pair. You end up having a molecule. Looks like it's got a hole in it, but it's more shaped like these blobs go together in more of a tetrahedral. Heme globin is an interesting molecule. After the first oxygen binds to the first heme group, it facilitates the binding of a second oxygen to another heme group, and then the third, and then the fourth. So each time an oxygen binds, the next oxygen binds more quickly. This is unusual in chemistry, usually when you have a system that does the same thing a bunch of times. The first instance is the easiest, and then it gets harder and harder. So I made a model. I brought a model in. This is a heme globin nitric oxide complex, 4N8T. And if we zoom in on it, what we see are the heme groups. It's a little bit limited in the color palette I could use. Sorry about that. The hemes each have a nitric oxide, but you can kind of see the bent geometry that an NO or a O2 would adopt. And this molecule was created by exposing regular heme globin to nitrite. And here we have two ions of nitrite, NO2 minus. There should not be a bond in between them, but essentially these nitrites occupy the center of this particular structure. An interesting feature of this structure is this NO, iron NO angle. It turns out from this study that it was found that the iron NO angle depends on how you introduce the NO. So there's two ways. One, you can take heme globin and allow nitric oxide NO gas to diffuse into it and you'll get reproducibly one angle iron NO. If you use this nitrite method to produce the nitric oxide, you get reproducibly a different bond angle there. And the bond angles are different because of the effect of the protein. And here you can see the different alpha and beta segments specifically. I think an alpha-beta pair would be orange and purple. I'm sorry, green and purple and another pair is yellow and blue. So the lesson to be learned from this protein is that what's happening at the metal center here, in this case the iron NO, is not something that can be reproduced without the protein being present. If you just take heme and do this chemistry, there's only one iron NO angle. You might also ask, is there interconversion of the two? I believe you have to heat up the protein past where it can decomposes to get these two to interconvert. But clearly one of them has to be a kinetic product and one of them has to be a thermodynamic product. Okay, going back to the top. So hydrogen bonding, as we said, is a thing. Myoglobin is another protein. Hemoglobin takes oxygen from your, collected in your lungs, passes it through the blood stream and hands it off to myoglobin. And myoglobin takes the oxygen from hemoglobin and distributes it within the tissues. Myoglobin basically looks like one of these subunits, like one of the colors. It's a smaller molecule than hemoglobin. The bonding in hemoglobin and myoglobin have to be just strong enough for the oxygen to make it from your lungs to your tissues and then be transferred to the myoglobin and then to allow the oxygen to escape and be used in metabolic processes. So it's all very finely balanced how strongly these things bond. Part of this is the hydrogen bonding. You can see there is ability of oxygen to hydrogen bond. Things like carbon monoxide, which is a poison for hemoglobin and myoglobin, carbon monoxide doesn't do the hydrogen bonding nearly as much. So, question that arises at this point, how do we know structure? Well one answer is actually crystallography and that's probably the most popular method, most common method. But there are other methods, neutron diffraction. Of course you need a neutron source like a reactor or something to do neutron diffraction. And a newer one called cryo electron microscopy on this latter one is really useful because it doesn't always require crystalline samples. And so I think cryo electron microscopy is going to be a technique that is going to be very useful in the future for determining protein structures, important protein structures, in cases where we can't get crystals. There's also magnetic resonance methods. And when you think magnetic resonance, often people think about imaging, but really I'm talking about more of the NMR technique, nuclear magnetic resonance. It's not radioactive or anything like that. It's just atoms have nuclei. So the word nuclear is just an adjective there. You can think of NMR methods as being radio wave phosphorescence phenomenon. Essentially you have a sample, you can hit it with a bunch of radio waves all at once and then for a few seconds afterwards you might detect. I was going to say here, but let's use the word detect, radio waves coming out. And from the intensity versus time plot of the radio waves that come out, you can infer after Fourier transform of course some details about the structure of compounds. For proteins you can do two-dimensional, three-dimensional methods way out of the scope of this talk, but you can figure out which atoms are close to each other and therefore deduce features of the structure. A few words about X-ray crystallography. It basically rests on interference patterns. I don't have Bragg's law up here, but Bragg's law is important. How this works? Think of an X-ray beam coming into a surface. And the X-ray beam could bounce off a top layer of a surface or a bottom layer of a surface. Well, in the incident light the X-ray beam, all of the waves are in phase with each other. As the two beams coming out of the sample travel different distances, they're no longer guaranteed to be in phase with each other. If they happen to be in phase with each other, you get the thing property called constructive interference. And then you will see a bright spot at some position where there's a large concentration of detectable X-rays. The other limiting type of interference is destructive interference where the peaks and the troughs line up. There we go. Where the peaks and the troughs line up. And so they cancel each other out, leaving dead space. You don't detect any X-rays. So essentially what happens with a crystal is a bit more complex. You can think of an X-ray beam coming in and then being diffracted from a crystal. Since a crystal is a three-dimensional diffraction grating, what you end up with is a three-dimensional pattern of spots around the crystal. And this pattern of spots depends on the angle the X-ray beam comes in. So essentially what we do is we do the experiment shown here. Take an X-ray beam, shoot it at a crystal and measure the positions and intensities of the spots in three-dimensional space. Then you rotate the crystal a little bit, do another measurement, and you might take several thousand of these measurements to build up a data set and from that data set you can deconvolute the map and make a model of whatever makes up that crystal. In this case, in this talk we're talking about proteins. So modern X-ray diffractometry. So essentially here's a single crystal X-ray diffractometer. There's a powder X-ray diffractometer. That's just what the instrumentation looks like. And the crystal growth part really deserves some mention here. It's an art to grow crystals, especially of proteins that are not guaranteed to give you crystals. I was able to find some nice photos from NASA. Here's Commander Alexander Gerst as he is, I think he is, setting up proteins to be crystallized in space. Crystallization under zero gravity conditions evidently works really, really well. Here are some crystals that were actually grown in space. And essentially the popular method used to grow protein crystals is essentially where you take like a drop of your sample and then you invert it over a small cup that's got a solution in it that will tend to capture water. So it's essentially like a drying agent. And you wouldn't just set up one of these little droplets. You might set up almost 100. This is a 96 well-plated container. You might set up 96 of these. What you want are little crystals, little single crystals, maybe 200 microns on the side. What you don't want are things that are twinned. Those types of crystals are harder to process the data. You can do it, especially with modern computers and the like, but it's just harder. Ideally, you just want one nice crystal. All it takes is one crystal for you to be able to get good data. What you do with the data, well, after processing you essentially have a snapshot of the molecular structure. You have a high quality crystal structure then you've got knowledge of where all of the atoms are. We have made tremendous advances in extra crystallography in the past three decades. Now, this is very useful when your other methods aren't enough. Sporting methods are nice, but it's obviously nicer to have like a snapshot. I don't want to give you the impression that having an extra crystal structure makes it definitive that you know what your compound is. There are a lot of assumptions that go into figuring out a structure and sometimes those assumptions are wrong and can give you results that need further interpretation. There are a number of publicly accessible databases for small molecules, for minerals, and for protein crystallography. Just about everything I talked to you about today is coming from the RCSB site. Once you've got the data, if you remember the public, you can use things like Jmol or Chimera or PyMol to actually view the data. Jmol, PyMol, Chimera, you can even just type in the identifier from the RCSBPBB and it'll just download the data by itself. Here's an example, the 4D1N and that's a neuronal nitric oxide synthase. What you get when you just open it up in Jmol is where all the atoms are and after some clicking around and some playing, you can get it to look like a nicer structure. As you can see, the view of every atom in the structure is not really necessary because if you represent some of the action happening in the structure by cartoons, then you can focus on what's really important. Basically, moving on to some other examples, heme thiolate proteins. I mentioned cysteine in the proximal site. Heme thiolate proteins are incredibly important. They do a variety of things, both in the body and commercially. And I was going to talk to you about just a couple. Cytochrome P450, I've already showed you that structure. The P450 comes from the wavelength of light, that the carbon monoxide complex of this thing shows. It basically got a maximum absorbance at that frequency. I'm also going to show you a nitric oxide synthase. Three things that these guys can do is they can put an oxygen on some substrate. And what we'll see for neuronal nitric oxide synthase is that the molecule will take an amino acid, L-arginine, and turn it into L-citrulline by breaking off a nitrogen and shoving an oxygen on it to make an anion nitric oxide. And there's plenty of other things that these molecules do. Okay, so cytochrome P450. You've got cytochrome P450s in you. They're in your liver. They help do things like detoxify organic compounds. For example, say you've been exposed to some benzene, benzene C6H6, a cytochrome P450 could take some benzene and turn it into phenol, which is C6H5OH. The OH group improves the solubility and allows your kidneys to filter out the molecule and even urinate it out. P450 is a very important molecule. And I think I've got examples of a cytochrome P450 somewhere. No, I have the horseradish peroxidase instead. Okay, so moving along. So one thing that we've seen before is compound one. Compound one is very highly oxidizing. And what it can do is grab a hydrogen atom from some other molecule to make essentially an OH. Well, when this happens, say your benzene turns into a phenyl radical after it loses its hydrogen atom. The phenyl radical is particularly reactive and will just make off with the whole OH from the iron and turn into phenol. This is why it's called a rebound mechanism because the consequence of iron oxo stealing a hydrogen from the substrate is that the substrate comes back and steals the whole OH. Very classic type of chemistry. Here we get into the neuronal nitric oxide synthase. This is a model of the NNOS that I have here. It's the 4D1N. And this one actually has a l-arginine mimic in the active site. Okay, so here's the heme. To save time in processing this structure, I left all of the cofactors, as they are called, since they're not part of the protein chain in orange. And there is a heme group over here. There is another heme group over here. There's the iron. And both of these subunits are identical. So we'll go back to the first one. In fact, there is this, you can see this longer chain out here that is poised above the iron atom of the active site. I'll also point out this is a tetrahydrobiopterin molecule. And it can store up to four hydrogen atoms, which means four H pluses and four electrons. And this would be part of the electron and proton transfer chain that would allow the heme group to work. The detail of this active site is reproduced over here. So what I have over here is an electrostatic map of the pocket of the heme. You can see the heme is in there. There's the iron atom. I made it shiny because I like shiny things. What you can see right here would be one of the carboxylate groups. Red indicates area of negative charge. I believe there's a hydrogen bond happening over here. Is it negative? No, I'm sorry. Red is positive charge. So that's why this hydrogen bond is happening. That's why the iron down here is red. This atom is supposed to be a sulfur atom from the phylate that is in the proximal site. And above you can see our L-arginine mimic. It binds more strongly than L-arginine does in this particular molecule because it's got a slightly changed end in the active site and then a more strongly hydrogen bonding at far from the active site. This would be a molecule that would deactivate nitric oxide synthase. And there's our tetrahydrobiopterin as well. And you can see some of the more spaghetti parts of the protein that are holding everything in place. One thing that is nice is that through the colors on the electrostatic map as molecules make their approach to the heme group, you can actually see how the molecules are directed to being in the right place at the right time. It's also an acetate down here from the crystallization conditions. In all of these structures I haven't shown explicitly the hydrogen atoms because that just adds complexity that doesn't help with seeing what the active sites are doing. Okay. Nitric oxide of course is very important. The Nobel Prize for Physiology or Medicine was given in 1998 for the discovery that nitric oxide has a biological role. It can do things like it's a neurotransmitter. It's a signaling agent that helps control physiological blood pressure. It's even secreted by white blood cells since it's a nasty molecule. It would act like bleach if your white blood cell throwing something that acts like bleach on a bacterium is probably part of your job description. So essentially it's produced in the body as I've been saying by l-arginine. In our mimic we had a ring up in this part instead of just the l-arginine. An l-arginine gets oxidized. There's an OH that attaches itself to one of those ends because of the action of the heme group. And then further along we basically remove a nitrogen. There we go. We remove a nitrogen and it comes off as NO. Notice how there's only two nitrogens in that unit where there used to be three over here. So this is just a two-dimensional representation of the active site with hydrogen bonding. So you can see what the pocket of the protein looks like on the inside. Just mentioned that's a dimer as well. There's a dimer of dimers. In fact there's a small subunit and a larger subunit and then those two dimerizes. And I've mentioned all of these things. So not only is NO produced by a heme-containing enzyme, it's sensed by a heme-containing enzyme. And once it's sensed it basically gives you guanodile cyclase. I think that's what that's called, which is another signaling molecule that downstream will cause the blood pressure to go down. Essentially there's a muscle tissue that surrounds arteries and it's called smooth muscle tissues. And when it receives the signal from nitric oxide to relax the blood pressure will go down because there's more space in the blood vessels. So what exactly does it do? Well it basically starts this cascade. One thing that nitric oxide does for blood pressure, when you think of drugs such as Viagra for example, that the action of Viagra has to do with blood pressure, with relaxing smooth muscle tissue or not relaxing smooth muscle tissue certain places in the body. And the more nitric oxide you've got in the right place at the right time, the more physiological effect it'll have. Well there's two ways of getting nitric oxide levels to increase. One way is to make more nitric oxide. The other way is to stop the nitric oxide that is being made from decomposing. So you know basically in the body there are physiological mechanisms that get rid of nitric oxide once it's done its job. And drug such as Viagra basically put a damper on the cycling or processing of nitric oxide. So basically it plugs the drain let's say and you know allows each NO to have a longer lasting effect. So some last thoughts hems really important. It's not the only cofactor that you find in the body but it's an important one. It has a lot of different functions in enzymes. You know essentially it's got a lot of flexibility for such a molecule. You know what's in this site number six as I'll call it the proximal site plays a huge role what the hydrogen bonding network does in the distal site as well as the proximal site plays a huge role in determining its chemistry. So you know basically this site here would allow you to do a 3D print of a heme molecule or hemoglobin protein. It's basically the files you would need if you had a printer and you know you could make yourself a little blob of hemoglobin. Okay so again acknowledgements I started out with acknowledgements. I've got some useful references here. I want to thank everyone for their attention. Again thank NSF and my school for their support. So essentially that's what I have for you today and I hope you've enjoyed today's talk.