Biochemistry 15 Doctor /7/2012

Transcription

1 Heme The Heme is a chemical structure that diffracts by light to give a red color. This chemical structure is introduced to more than one protein. So, a protein containing this heme will appear red in color after being exposed to light, i.e.; Hemoglobin. Heme s basic Chemical Structure: It is composed of four five- membered Nitrogen rings. Those rings are interconnected by ( - CH= ). Interconnections occur at the ring carbons, which are ortho in position to Nitrogen. This interconnection is responsible for forming a larger ring called Protoporphyrin ring. (Aka: Porphyrin ring). Porphyrin is the large ring without including the Iron atom in the center. Iron (Fe) is a transition metal that can make up to 6 bonds. If iron comes in the center of the porphyrin ring it attaches to the four nitrogen atoms. - Why do physicians administer Fe supplements for patients suffering from iron deficiency anemia? When a patient takes iron supplements, [Fe] will increase in the body. This increase stimulates the body to synthesize more porphyrin rings in order to bind to the high concentrated Fe atoms. This binding forms more heme thus increasing [Heme]. This increase stimulates the body to synthesize Globin protein (a four polypeptide chained protein). Heme then attaches to Globin forming more Hemoglobin, which increases its concentration. Consequently, the Oxygen carrying capacity will increase, treating anemia. Types of Heme There are a lot of types of heme. All of them have the same basic structure in common (a porphyrin ring + central Fe atom). They only differ in the attached side chains. Most of the hemes attach to proteins via non- covalent bonds Hydrophobic interactions (this occurs in the hydrophobic pockets). Heme C is a special type of heme. It binds to 10 Cys residues thus making a covalent bond with the protein. Recall that Fe atom can makeup to 6 bonds. Four of them are already engaged with the nitrogen atoms of the four rings. Two binding sites are left, one up & one down. Rule of Coordination Ligation : * 4 bonds = 4- coordinated * 5 bonds = 5- coordinated the most abundant * 6 bonds = 6- coordinated Generally, most of the hemes that are found within are 5- coordinated (that is, it forms 5 bonds, four with the nitrogens & one with an amino acid. The 6 th binding site is left free). Amino acids that can bind to Fe include: Lys, His, Met. Q: Does Fe have a charge on it? A: Yes it has. This depends on the attached group. This depends on the type of the attached group whether it is a reducing or oxidizing group. That is to say, Fe can be oxidized or Reduced (Fe can form Fe +1, Fe +2, Fe +3, so it can gain more than 1 electron). This is the exact effect of heme on the protein especially in the electron transport hemes (they can change between Fe +2 & Fe +3 ). 5- coordinated - > Fe coordinated - > Sometimes Fe +2, and it can be Fe +2 1

2 Function of Hemes There are numerous functions. They depend mainly on the types of attached groups on Fe and the type of coordination (ligation). For example, the Fe in Hemoglobin is 5- coordinated; four bonds with the nitrogens, one with an amino acid, and the last one is opened for the attachment of O2, CO2, etc. Some of heme functions include: O2 activation, O2 transfer, and electron transfer. Heme that is involved in electron transfer is 6- coordinated because it does not need a binding site for oxygen. It only acts as a medium to pass electrons. Heme that is involved in Oxygen activation is 5- coordinated so that the 6 th place is free for binding with O2. Nevertheless, some are 6- coordinated, but when the heme is reduced the ligand detaches from the 6 th place to allow for the attachment of Oxygen. This is known as the Dynamics of Proteins. Myoglobin Myoglobin is a protein that is found in tertiary structure. It was the first protein to be determined structurally using X- ray crystallography. It is composed of eight α- helices and no β- sheets. It is formed of 153 amino acids that form a single polypeptide chain. It works as storage for Oxygen. Myoglobin s hydrophobic amino acids are directed to the interior of the protein. On the other hand, Its hydrophilic amino acids are directed to the exterior of the protein. Heme in Myoglobin: It is 5- coordinated; four bonds are the standard ones (connected to Nitrogens of the Porphyrin ring), the 5 th is with His, and the 6 th place is free for binding with the ligand, i.e.; O2, CO2. The affinity of heme to CO is about times more than O2. Q: What can be found in the hydrophobic pocket? A: The residue His (other than the one that could be bound to the heme). When Oxygen binds to the heme, it binds straightly. This orientation causes repulsion between the Oxygen itself and the residue His. This will happen because there is no enough space for both of them. So, Oxygen will be bent (angulated). Q: What is the effect of the presence of the residue His (in the pocket) on the function? A: The presence of His makes the bond angulated so as to lower the affinity of heme to O2 & CO2. Do we need the affinity to Oxygen to be high or low? Actually it depends on the type of protein and its main function. Recall that Oxygen will not bind to heme forever. Instead, it is transferred (Attach Detach). For example, you need the affinity to be higher in Myoglobin than in Hemoglobin because Myoglobin is found in muscles as storage of Oxygen. When the muscle needs it, it is provided by the Myoglobin. On the other hand, Hemoglobin transfers Oxygen from lungs to tissues through blood. So, if the affinity in Hemoglobin is high, it will not be possible to release Oxygen to the tissues. Consequently, the affinity must be low enough in order to make hemoglobin affected by surrounding conditions (will be discussed later in this sheet) and release oxygen to tissues. 2

3 If there is no His, there will be high affinity, which means high capability of binding but low capability of detaching. Q: What is the function of His in the pocket? A: Lowering the protein s affinity for Oxygen by angulating the Fe- O=O bond. Hemoglobin (Hb) Hemoglobin is another protein that is similar to Myoglobin. It is composed of four polypeptide chains rather than one. Two of those chains are called α subunits (identical) and other two chains are called β subunits (identical). α subunits = formed of 141 aa. β subunits = formed of 153 aa. Each chain has a hydrophobic pocket to which the heme attaches. Heme binds to Oxygen. Q: How many Oxygen molecules can one molecule of Hb bind to? A: Four molecules, because Hb is composed of 4 peptide chains, each chain has a hydrophobic pocket that contains one heme that can bind to one Oxygen molecules. Recall that Fe in each heme is 5- coordinated. Myoglobin vs. Hemoglobin The way that Oxygen binds to Hb is different from the way it binds to Myoglobin. Why is that? It is due to the function difference between Myoglobin and Hb. Myoglobin is mainly an Oxygen- Storing protein whereas Hb is an Oxygen- Transferring protein. Logically, the affinity to oxygen cannot be the same. As mentioned earlier, the affinity to Oxygen in Myoglobin is higher because it stores it. When there is an excess of Oxygen, the percentage of saturation can be determined to either two proteins. This can be accomplished to Myoglobin by exposing it to Oxygen. When Oxygen reaches a pressure of 1 torr, it is said to be a 50% saturated Myoglobin. 50% saturated Myoglobin means that 50% of the already existing Myoglobin molecules are fully saturated with Oxygen. When increasing the pressure, the level of saturation decreases gradually (it becomes harder for Oxygen to bind to the protein). This is because the random movement of the two molecules. Explanation: Before reaching the 50% saturation, most of the binding sites at the protein are free, so the number of successful collisions is high. By passing the 50% the number of free binding sites decreases. Consequently, the number of successful collisions will decrease. Hemoglobin works a little bit different. It works by the principle of Positive Cooperativity. In Hb, the first oxygen molecule that binds to the first heme makes a conformational change in the protein structure so as to facilitate the binding of the second Oxygen molecule to the second heme. The second heme in turn acts the same to facilitate the binding of the third Oxygen molecule to the third heme and so on until all the binding sites of the 4 subunits of Hb are occupied by Oxygen. When the 4 hemes are oxygenated, the Hb is said to be 100% saturated. Q: How can the oxygenated heme facilitate the oxygenation of the following heme? A: Bu changing the protein s conformational structure. 3

4 Positive cooperativity means that the affinity to Oxygen is low at the beginning, but it increases due to the facilitation of oxygenation caused by a conformational change. Q: When does Hb reach a 50% saturation level? A: When only 2 hemes are bound to Oxygen (oxygenated). Q: At what pressure the Hb is 50% saturated? A: At 26 torr If it is not possible for Hb to reach the 50% saturation level at 26 torr, then the affinity to Oxygen will be low. So, it is easier for Hb to lose Oxygen rather than gaining it. In lungs, The pressure of Oxygen is about 100 torr, which is enough for the 100% saturation of Hb. However, when Hb reaches tissues (which have a pressure of 20 torr), it becomes pretty much easier for it to lose Oxygen. This is because 20 torr is less than the 26 torr (the 50% saturation pressure). Slide 5; (A) represents a deoxygenated Hb, which appears more relaxed, more opened. (B) represents an oxygenated Hb, which appears tighter, more closed. ph- Hb Relationship Bohr s Effect Tissues have metabolic activity by default. This process increases the acidity due to the increase in [Lactate] which increases [H + ]. As a result, tissues are more acidic than lungs. High [H + ] makes all the amino acids that can protonate do so (high [H + ] prevents the amino acids from losing their protons because there is already high amount of H +. Q: When does the salt bridge appear? A: At high acidity (especially in tissues). This is exactly what we need in tissues the low affinity to Oxygen so it can detach easily and be supplied to the tissue. Q: How does CO 2 travel in blood? A: It travels as a buffer (H2CO3/HCO3 - ). The transformation of CO2 to (HCO3 - + H + ) in blood lowers the ph thus lowering the affinity to Oxygen(lowering the percentage of oxygenation) and vice versa. O 2 - CO 2 Exchange When the tissue produces CO2 and it converts to H2CO3, it lowers the ph in the tissue thus lowering the affinity to Oxygen. This will release new Oxygen to the tissue to continue its metabolic activities. BPG BPG is 2,3- BisPhosphoGlycerate. It is found in blood. BPG in general is negatively charged because of the presence of phosphate groups in addition to CO2 -. It has a specific place at Hb for binding. When the negatively charged BPG is bound to Hb, it forms a sort of electrostatic attractions with the positively charged amino acids, namely His 143. For example, When His 146 is protonated, it is positively charged, and so it can bind to a negatively charged amino acid making a Salt Bridge. The salt bridge changes the conformation of protein thus lowering the Oxygen affinity. 4

5 Q: What is the difference in structure between adult Hb and fetal Hb? How can this difference affect the function? A: Structure => Adult Hb is composed of 2α subunits due to the activity of α producing gene, and 2β subunits due to the activity of β producing gene. Fetal Hb is composed of 2α subunits but NO β. Instead, it has 2γ subunits due to the activity of γ producing gene, which is not found in adults. β is similar to γ except that there is a small difference in specific amino acids, thus affecting the function. Function => In adult Hb, His 143 is present in β subunits. It can interact with BPG. Consequently, it lowers the affinity to Oxygen. In fetal Hb, however, NO His 143 is present. Instead there is Ser. Recall that Ser is not positively charged and so it cannot interact with BPG. This prevents the effect of BPG. SO, the affinity to Oxygen is NOT affected by BPG. Conclusion: - At all time, the fetal Hb has higher affinity to Oxygen than the maternal Hb. This is why the fetus always takes Oxygen from his mother. - Maternal Hb; α2β2 - > His > BPG effect. - Fetal Hb; α2γ2 - > Ser - > No BPG effect. Collagen Collagen is a protein that is composed of 3 α- helices that are twisted around each other. This twist creates a sort of nodes called site of twisting, every 3 amino acids. At each node in each α- helix, the side chain of the existing amino acid that is, the third amino acid in sequence is directed interiorly. Since there is no enough space at the node for the side chain, it must be as small as possible. The simplest side chain goes for Gly, which will be the amino acid at the node. The third amino acid in sequence means: the multiples of 3. 3 rd aa, 6 th aa, 9 th aa, etc. Twisting results in forming ProtoCollagen Ser Met Gly Arn Val Gly Ala Ser Gly Glu Ser Gly Asp Ser Gly Val Glu Gly Met Lys Gly Ala Arn Gly Met Lys Gly 1 st 2 nd 3 rd 4 th 5 th 6 th 7 th 8 th 9 th Collagen has high amount of Proline, Hydroxyproline, Lysine, and Hydroxylysine. Q: Why is the existence of Hydroxyproline and Hydroxylysine important? A: Because both of them can make cross- links. After twisting the α- helices, they have to be cross- linked to each other. This is majorly done by Pro, ProOH, Lys, LysOH. The cross- linking is the major contributor in the tensile strength of Collagen (this is what we exactly need from collagen to be). There are some diseases that cause the collagen to be weak due to the deprivation of cross- linking. The major and most common diseases are Scurvy, and Osteogenesis imperfecta. 5

6 Scurvy A vitamin C deficiency, which leads to the inability for certain enzymes to Hydroxylize the amino acids Pro, Lys. This prevents the formation of cross- linking. The formed protein (Collagen) will be weak. Patients suffering from Scurvy are subjected to the rupture of blood vessels (because the walls of blood vessels are rich in collagen), thus suffering from bleeding. Prognosis: Bruises under the skin, Bleeding gums, Loose teeth. Osteogenesis Imperfecta A genetic mutation affecting the enzymes that can make hydroxylation. Cross- links do not for. Consequently, the formed Collagen will be weak. The result is a very weak and soft bone. It mainly affects collagen that is present bone (Recall that bone is rich in collagen). Patients suffering from this disease are subjected to bone fracture. They may also encounter bluish skin because it is showing the blood vessels underneath it. Types of Collagen There are about 28 types of collagens up- to- date. The major types in the body are Type I, II, III, VI, V. They are distributed throughout the body depending on their degree of cross- linking, Thickness, Strength to suit the tissue they are found in. Refer to Slide#8 for further details. It is very important. Keratin Another protein that is found in hair, fingernails. It might be sold in the markets. Structure 2 α- helices twisted around each other forming the Coiled Coil Shape. If two coiled coil segments twist around each other, they form the Proto- Filament shape. If four proto- filament segments twist around each other, they form the higher structural level, the Filament. The more the Cys content in Keratin, the harder it will be. This is because Cys forms disulfide bridges. So, the higher amount of Cys, the higher number of disulfide bridges. Some people apply chemical substance on their hair in order to break the disulfide bridges and soften their hair. Elastin Elastin is rich in Hydrophobic molecules; Gly, Val, Pro. It contains mobile hydrophobic regions where Lys residues are cross- linked. Recall that Pro and Lys cannot make cross- links without being hydroxylized. So, Elastin does contain Hydroxylysine (LysOH). Hydroxylation of Lys occurs by an enzyme called: Lysyl- Hydroxylase. With aging, the elasticity of tissues decreases underneath the skin, and so Wrinkles appear (mainly in face). It is very important to refer to slides; they contain information that is not mentioned in this sheet. By: Yazeed Lu ai Abdulla. 6

7 7