 Hello, welcome back to metals in biology. Today we are going to discuss control and use of metal ion concentrations. So, we need to control the metal ion concentration, not only we need to ensure metal ions reaches their destination, but if a lot of metal ions are getting accumulated at a given site, that could also be potentially extremely harmful. Of course, deficiency of metal ions can also lead to many disease states. So, it is essential therefore, to control and use of metal ion concentration for proper functioning of our biological systems. To maintain a particular metal ion in proper range, the term which is usually we use called homeostasis. Of course, one need to remove excess or unnatural metal ions otherwise it would lead to the toxification. So, detoxification is essentially an important process which removes excess or unnatural metal ions. Now, to remove these different metal ions, one can think of using extra cellular carriers. Of course, there could be passive transport, active transport in the form of ion channels and pumps which we will discuss in subsequent few courses. Now, there are metalloregulation that also are required. So, metalloregulatory protein will control the concentration of metal ion. Once again, homeostasis and detoxification are two important subtopic that we must understand. Detoxification becomes essential because otherwise it can metal ions can lead to different disease state. There are as I mentioned, there are different ways by which metal ions can be removed from a particular site. Active transport, passive transport, there are metalloregulatory protein that also control metal ion concentration. Binding and release of metal ions to receptors are controlled by pH and redox changes. So, for example, you know iron 2 plus versus iron 3 plus oxidation state change will control the binding of this particular metal with a particular ligand. For example, some of the sites are iron 3 plus specific because let us say too many anionic ligands are there. In that particular site, iron 2 plus is not a good fit. We will discuss one such case today, but once again the control of a metal ion and their usage must be done in a systematic fashion. Redox plays a crucial role to control any redox active metal ion concentration. Obviously, at given pH will be also critical for controlling the metal ion concentration. Well, we will discuss in subsequent slide the metal ion concentration control and how they are affecting different activities. In the last class, we are essentially discussing this cyderophore entrobactin where we have seen in bacteria how a particular metal ion we were discussing in particular iron centers, how iron centers or iron ions are getting accumulated inside bacteria by citric acid or by entrobactin. Today, we will see one such internalization process by mammals let us say, let us say in humans. So, how iron centers gets accumulated in our body that is what one of the subtopic we will discuss today. Now, these the transferrin is the protein that is involved in accumulating iron sites. Transferrin has 2 subunit, it is a huge glycoprotein it has 80 kilo Dalton molecular weight and it binds iron 3 plus very strongly ok. And what is found that the protein binding site of iron are also capable of binding carbonate CO 3 2 minus. Both iron 3 plus and carbonate CO 3 2 minus are binding to the transferrin synergistically. Of course, perhaps you know the metal containing protein when the metal is removed that is called apoprotein. So, apotransferrin is the one where iron site is not there and also cofactors such as in this case CO 3 2 minus is also missing. Now, this transferrin has 2 protein domains. In each domain, there are 2 subdomains that clamp down on the iron and carbonate ions. Once again iron 3 plus and CO 3 2 minus bind synergistically to the apotransferrin. Let us look at the transferrin structure. So, on the left we have the apotransferrin where iron is missing. Now, during this process of iron binding these are the 2 subdomain. This is one subdomain, this is another subdomain. These 2 subdomain are linked while iron is binding to this overall apotransferrin to give the iron transferrin protein, then essentially also carbonate comes into picture and they bind synergistically. Both iron and carbonate binds together in this whole protein overall to form a iron transferrin complex. Now, one thing you must have noticed the different the distance between these 2 subunits are decreased significantly during the iron carbonate binding. So, this is what is called the hinge motion. Hinge motion accompanies when iron carbonate binding are happening. So, this is similar to the hinges we know that it clamps down together ok. These 2 things are clamping down on the iron or carbonate indirectly overall iron and carbonate bringing these 2 subdomain closer together. Look at the distance between these 2 sides and look at the distance between here. Now, as we mentioned this is a huge glycoprotein. We are able to now see how iron and carbonate binds with each other. Now, these transferring active site geometry if we zoom in, if we zoom in here then we will be able to see perhaps the coordination complex inorganic metal complex that is forming when iron and carbonate is binding to the apotransferrin over here. Let us look at the transparent active site geometry. The crystal structure we have and that is fantastic to have because that gives you a crystal clear information about the active site. So, we find in the crystal structure that iron site which is over here iron site is binding with one histidine, one tyrosine and another tyrosine over there. So, 2 tyrosine and 1 histidine is bound with the iron site along with an aspartate. Over here this is the carbonate CO3 2 minus. So, both iron and carbonate are binding together with 2 tyrosine and one histidine and an aspartate bound iron center. So, this iron center is hexacoordinated. Quite interestingly and of course, this is a crystal structure obtained from lactoferrin. Quite interestingly you see that an arginine is present this is the arginine which is not coordinated directly with the metal center, but this arginine is present overall in this active site. An arginine active site forms a key hydrogen bond with the coordinated carbonate ion. This is the carbonate ion as you can see there is this hydrogen bonding with this carbonate ion. Therefore, overall it helps to effect protein folding around the metal coordination sphere. So, you have seen the iron center carbonate how carbonate is also hydrogen bonded with the arginine. Now, these 2 sub domain that we were talking about in the apotransferrin they bind with this iron and carbonate and both the sub domain comes close to each other as if they are clamping down on iron and carbonate. So, this crystal structure is quite informative in understanding how iron is binding with these apotransferrin to make the iron transferring and this gives us a overall picture crystal clear and very molecular level detailed picture how these are transporting or helping in internalizing the iron center in human. Of course, these carbonate that we have seen over there is not the only carboxylate ligase and present in metalloproteins. There are other carboxylate ligase and in metalloproteins and those are quite also interesting. So, let us look at the biologically available biologically available carboxylates, biologically available carboxylates. Well, one since we have seen carbonate another easy one would be the bicarbonate right. So, we have bicarbonate of course, another important one could be the aspartate one right. So, of course, I am not drawing the stereochemistry correctly here or CO2 minus and NH3 plus aspartate right. So, this is aspartate ASP aspartate and it also has these carboxylate ligation or it can provide carboxylate ligation in metalloproteins. There are other amino acids which are essentially of the similar type such as glutamate. You can also have once again the stereochemistry on this center I am not drawing. So, this is glutamate glue. We can also have lysine carbamate. So, where we have aspartate glutamate and lysine carbamate where we can also have the similar binding of this carboxylate ligation in metalloprotein. So, here we will have NH, CH2 and CH2, CH2, CH2 and then of course, carboxylate ammonia and H. So, this is lysine carbamate or carbonate is encountered in transparent as you have seen. This last one is found this carboxylate over here is found in urease, rubisco and phosphotryesterase right. There are different sites where different of these carboxylate ligation can be found. We might will be seeing quite often this aspartate, this glutamate and sometime even the bicarbonate. Well, it is also important to note that we are also having in addition to the carbonate binding that we were showing. Essentially other anions such as phosphate, arsenate, pyrophosphate, citrate, oxalate are all capable of binding with the metal center. There are different enzymes which can also bind or metalloenzymes that can also bind this sort of anion. Of course, ferric binding protein can also bind these phosphate, arsenate, citrate, oxalates, pyrophosphates. Those studies are done quite essentially quite a lot. What it can be concluded that not only carbonate various anions can also bind transferring in bacteria, which also have a transferring receptor. Well, another thing that becomes very clear that iron must bind as iron 3. This is iron 3 plus not iron 2 plus. So, this has to be in the ferric state. If it is reduced then of course, you need one need a bacterial reductase right. Thus affording the control of iron binding and uptake in the organism. So, this site for example, is very good in only specifically binding iron 3 plus, but this will not be a great binding site for iron 2 plus. Overall therefore, we can have control of iron binding particularly even iron 3 plus center right not for iron 2 plus center. And this sort of control binding and very selective binding also helps in uptake in the organisms. Now, let us look at how human transferring is actually involved in the iron release. So, how is overall process happening? So, the iron-free apotransferring that is the first step that we look at. This is each of the centers can load one of the iron. These are two different sub domain. Now, this iron-free transparent which looks like Peckman right. Now, it can pick up iron iron as you can see these are iron loaded transparent from outside the cell to inside the cell we are trying to see how iron is getting transported and what are the process involved. So, it starts with iron-free apotransferring. Once of course, there are receptor on the cell membrane and this receptor gets activated when iron ion is bound with the apotransferring. Subsequently, these receptors bind with this iron loaded transparent and they try to enter the cell. Overall a coated pit formation is happening. These are the clathrin which actually protects the vesicle formation or helps in the overall vesicle formation. Once this vesicle is formed, this is a coated vesicle as you can see from outside there was no such vesicle or protected information or protected apotransferring was there only after iron loaded transparent formation and getting activated and binding with the receptor one can then can see that this coated pit comes into the picture coated vesicle essentially it protects these are the fat layer right. It protects the iron center from getting misplaced so that it can reach to its target very reliably. Subsequently, once the target is kind of identified or target is reached, then uncoating of this you know of this coat that is nothing, but fat layer occurs. Now, this uncoating gives rise to a state where these iron can be released of course, it is going to be a going to be a ATP driven process and release of iron overall happens and then the rupture of this vesicle gives rise to the release of iron inside the cell and here in the ribosome so here these irons gets accumulated into the ferritin ok. We will come back to the ferritin in a moment. As you have seen transparent is the transporter of iron ferritin is the one which is responsible for storing iron inside the cell. Once it releases iron then remaining apot form of the transparent goes out of the cell membrane and from the cell overall again the catalytic cycle or the iron loading circle starts again. So, this is the human transferring mechanism how iron is getting released. Now, this iron release in cells by receptor mediated process is happening of course, as you have seen this is the receptor and this is happening you know in quite a reliable way it can be done again and again and again without any loss of activity as such. So, these are very effective process and that is the beauty of the biological processes. So, far what we have seen is how transparent is capable of internalizing or bringing the metal ions in particular iron that is iron specific iron is getting incorporated inside the cell. That is quite interesting it is a very simple process, but then understanding at a molecular level is quite exciting. As you have seen that there is a iron 3 plus ion not iron 2 plus and a carbonate that clamps down the 2 sub domain of apotransferring to bring them together. On top of that you have seen that the coordination site is quite interesting in these cases. We have 1 histidine and 2 tyrosine and 1 aspartate bound with the iron center along with carbonate. There is arginine is also patting or giving some sort of support through hydrogen bonding just giving the Koji feeling. So, that carbonate feels like welcome over there it binds with the iron strongly, but still further these secondary coordination sphere type of interaction through hydrogen bonding helps quite a lot in overall stabilizing these iron carbonate structure or the geometry in the apotransferring to give the iron loaded transfer. As you have also seen how these apotransferring are loaded and then they are getting carried inside the cell these are schematic diagram overall it helps in releasing the iron at a destination or it can be stored as a ferritin or in ferritin in multiple iron center can be seen stored. As you will see in this ferritin this sort of ferritin protein we can have even up to 1000 iron center that is quite exciting to note. And once again it is not only the carbonate that can bind with iron center or any other given center there are other carboxylate ligation in metalloprotein we have seen bicarbonate, aspartate, glutamate and lysine carbamate which are once again found in different active sites. Iron must bind as iron 3 plus in this transferring or apotransferring not in iron 2 plus state. So, the oxidation state plays a crucial role in controlling the metal ions and overall uptake in the organism right. Now, let us try to see the metal regulation of gene expression. As you have seen how metal ions is getting internalized in the cell now we need to also understand that we need to regulate the metal ion concentration inside the cell. Now, this metal regulation are governed by many different factor of course, without proper regulation of the metal ion concentration as we were discussing briefly that there are many consequences many diseases both higher concentration and lower concentration of metal ions can lead to many different diseases. Now, the principles behind these metal regulations are quite simple. Metal mediated protein structure changes affect transcription right. So, when metal is binding with protein the structural changes that happens by such metal binding with protein it affects transcription. Well as you know regulation of metal ion concentrations are going to be dependent both by transcription and translation let us just briefly mention what is translation and transcription as you all know perhaps. So, DNA is being converted to mRNA by transcription and the translation is essentially is the mRNA to protein formation right. So, overall so, DNA to mRNA formation and mRNA to protein formation these are the transcription and the translation and we will be seeing more of that in the next class. But let me try to tell you that metal regulation is quite an important topic which will determine the importance of metal and any milk malfunction in the regulation process can lead to the different disease. Metal mediated protein structure changes can affect transcription. Metal mediated protein structure changes also affect translation. APO versus hollow metalloproteins bind DNA and RNA completely differently. Of course, metallonegatory protein is a sensor just like what we have seen in different cases in inorganic chemistry everywhere metallonegatory proteins is the sensor. Metal induced protein structure changes also activate enzymes. So, if there is a metalloenzymes which does not have metal that means, the APO metalloenzyme. Now, once you incorporate the metal it is going to be active. So, metal induced protein structure changes during the metal internalization into the apoprotein. We are going to see the structure and changes and therefore, it participate also in activating the enzyme because these are metal enzymes we are talking about. Also metal induced protein structure changes are metal specific. Let us say there is a iron specific or iron specific enzyme it is not possible to induce the similar changes with any other metal. So, let me try to tell you once again that metal mediated protein structure changes affect transcription. Metal mediated protein structure changes affect translation. We will discuss this in little more detail. Metal regulatory protein is a sensor right, it senses what to do, it decides what to do or it tells what to do. Metal induced protein structure changes also activate the enzymes. Well we will see some of these processes in the next class. Let us keep studying and we will see how metal ions overall are involved in controlling the metal ion concentration as well as how they are getting stored in different places such as ferritin. Till then keep studying we will get back in the next class. Thank you very much.