Lecture 4 - Hemoglobin - Biochemistry I Fall 2024 PDF

Lecture 4 – Hemoglobin MED204 – Biochemistry I Fall 2024 Course Lead: Dr Chloe Antoniou, PhD 1  Oxygen carrying proteins Hemoglobin the most abundant protein in red blood cells efficiently carrying oxygen from the lungs to...

Lecture 4 – Hemoglobin MED204 – Biochemistry I Fall 2024 Course Lead: Dr Chloe Antoniou, PhD 1  Oxygen carrying proteins Hemoglobin the most abundant protein in red blood cells efficiently carrying oxygen from the lungs to the tissues while also contributing to the transport of carbon dioxide and hydrogen ions back to the lungs. Myoglobin is located in muscle and provides a reserve supply of oxygen available in time of need. Both proteins can bind oxygen because they contain a prosthetic group called heme (or haem). 2  The heme group Heme consists of a complex organic ring structure, protoporphyrin, to which is bound a single iron atom in its ferrous (Fe2+) state. understand how the hemo group looks like The heme-bound iron can be in either the ferrous (Fe2+) or ferric (Fe3+) oxidation state, only the Fe2+ state is capable of binding oxygen. The general structure of The heme group of a porphyrin ring hemoglobin 3  Oxygen binding to heme The iron ion can form two additional bonds, one on each side of the heme plane. These binding sites are called the 5th and 6th coordination sites. The 5th site is occupied by the imidazole ring of a histidine residue from the protein. This histidine is referred to as the proximal histidine. Oxygen binding occurs at the 6th coordination site. Oxygen binding changes the magnetic properties of hemoglobin which are the basis for functional magnetic resonance imaging (fMRI), one of the most powerful methods for examining brain function. 4 The structure of myoglobin His E7 is the distal His. His F8 is the proximal His. 5  Principles of ligand binding In general, the reversible binding of a protein (P) to a ligand (L) can be described by a simple equilibrium expression: The reaction is characterized by an equilibrium constant, Ka, such that: 6  The fraction q is the number of ligand binding sites on the protein that are occupied by ligand: For O2 binding to myoglobin: When [L] = Kd , half of the ligand-binding sites are occupied. 7 Oxygen binding to myoglobin understand the curves and their levels of complexity 8  Myoglobin vs Hemoglobin Myoglobin (Mb) and hemoglobin (Hb) are two evolutionarily related proteins that employ nearly identical structures for oxygen binding. Mb exists as a single polypeptide, whereas hemoglobin has four polypeptide chains, two a-subunits and two b-subunits. Myoglobin Hemoglobin 9  Sequence and structural homology between Mb, Hba and Hbb. Hemoglobin subunits have similar structure to myoglobin with a high amount of sequence identity and similarity. 10 Hemoglobin subunits form tetramers. In hemoglobin, the subunits interact with one another to form the proteins quaternary structure. These interactions are not possible when the sequences of the subunits are those of myoglobin so myoglobin is a monomer. 11 Oxygen binding to hemoglobin A sigmoid binding curve indicates that the binding of O2 at one site within the hemoglobin tetramer increases the likelihood that oxygen binds at the remaining unoccupied sites. Conversely, the unloading of oxygen at know the effects og CO and pH one heme facilitates the unloading of oxygen at the others. This sort of binding behavior is referred to as cooperative. Hb is an allosteric protein. Allosteric proteins are ones which the binding of a ligand to one site affects the binding properties of another site on the same protein. 12 Cooperativity enhances oxygen delivery by hemoglobin. 13 Hemoglobin is very effective in providing oxygen to exercising tissues. 14  Hemoglobin undergoes a structural change upon O2 binding There are two major conformations of Hb: the R (relaxed) and the T (tense) state. O2 can bind Hb in both conformations but it has significantly higher affinity of Hb in the R state. The R state is the predominant structure of oxy-Hb. The T-state is the predominant form of the deoxy-Hb. 15  Effects of carbon monoxide (CO) on hemoglobin. Binds at the same binding site as O2 to form carboxyhemoglobin (COHb). Binding of CO is 200 times tighter than that of O2. High concentrations of CO can be rapidly fatal (CO poisoning). Normal levels of COHb are 1-2%. The COHb level in smokers is generally in the 3–5% range. As a general rule, for each pack of cigarettes smoked per day, the COHb rises approximately 2.5% (Hampson et al., 2012). 16 Effects of carbon dioxide (CO2) on hemoglobin. High CO2 levels decrease the blood pH by a reaction catalyzed by carbonic anhydrase. CO2 is an allosteric effector of Hb. Both the presence of CO2 and the drop in pH affect oxygen binding to Hb. 17 The effect of pH on O2 binding to Hb 18  The effect of H+ on O2 binding to Hb Both O2 and H+ are bound by Hb, but with inverse affinity. →When the O2 concentration is high, as in the lungs, Hb binds O2 and releases H+. →When the O2 concentration is low, as in the peripheral tissues, H+ is bound and O2 is released. H+ helps stabilize deoxyhemoglobin in the T state by contributing to interactions such as that shown on the right. 19 The effect of CO2 and pH on the O2 binding to Hb CO2 reacts with the terminal amino groups of Hb subunits to form carbamate groups, which are negatively charged, in contrast with the neutral or positive charges on the free amino groups. The amino termini lie at the interface between the ab dimers, and these negatively charged carbamate groups participate in salt-bridge interactions that stabilize the T state, favoring the release of oxygen. 20  The Bohr effect The effect of pH and CO2 concentration on the binding and release of O2 by Hb is called the Bohr effect , after Christian Bohr, the Danish physiologist who described this in 1904. 21 Oxygen binding to Hb is regulated by 2,3-bisphosphoglycerate (2,3-BPG) 2,3-BPG is known to greatly reduce the affinity of Hb for O2. 2,3-BPG is important in the physiological adaptation to the lower pO2 at high altitudes. The 2,3-BPG concentration in erythrocytes also increases in people suffering from hypoxia. 2,3-BPG 22 23  Fetal Hb has higher affinity for O2 Fetal Hb consists of 2 a-chains and 2 g- chains. The g-chain and b-chain are 74% identical but differ in some key residues, leading to a higher O2 affinity to fetal Hb. One noteworthy change is H143S in the b chain, part of the 2,3-BPG-binding site and reduces the affinity of 2,3-BPG for fetal Hb. This difference in O2 affinity allows O2 to be effectively transferred from maternal to fetal red blood cells. 24  Temporal expressions of globin chains Image from Fontana L, et al., 2023 25 26  Hemoglobinopathies A group of inherited disorders in which there is abnormal production or structure of the hemoglobin proteins 1. Sickle-cell anemia An autosomal recessive disorder that results from the aggregation of mutated deoxyhemoglobin molecules. Vernon Ingram demonstrated in 1956 that a single amino acid substitution in the β chain (E6V) of hemoglobin is responsible. The introduction of a hydrophobic residue allows interaction of this part of the protein with another hydrophobic patch and initiates aggregation. 27 2. Thalassemia An autosomal recessive disease caused by the loss or substantial reduction of a single hemoglobin chain. In α-thalassemia, the tetramer consists of β-subunits only. These tetramers, bind oxygen with high affinity and no cooperativity. Thus, oxygen release in the tissues is poor. In β-thalassemia, the absence of β chains causes the α chains to form insoluble aggregates leading to red blood cell loss and anemia. The most severe form of β-thalassemia is called thalassemia major or Cooley anemia or Mediterranean anemia 28  Summary of what you should know Differences and similarities between Mb and Hb How CO, CO2 and 2,3-BPG affect the oxygen binding capacity of Hb Be able to answer questions when you are given an oxygen binding curve and you are asked about affinity, P50 and release of oxygen 29 The whole is more than the sum of its parts Aristotle 30