Hemoglobin and the Heme Group:
Inorganic Synthesis Experiment
Authors: Rachel Casiday and Regina FreyRevised by: C. Markham, A. Manglik, K. Castillo, K. Mao, and R. Frey
Department of Chemistry, Washington University
St. Louis, MO 63130
All of our specialized organs are united by their fundamental need for a particular chemical environment that will enable the body's metabolic reactions. This environment must supply oxygen and nutrients to furnish the building blocks for cells, enable biochemical reactions, and provide energy for the body. Additionally, it must be able to eliminate the waste products of the body's metabolic activities.
Blood distributes oxygen and nutrients to the many different cells in the body, carries CO2 generated by the cells to the lungs for exhalation, and carries other waste products to the kidneys and liver for processing and elimination. Many finely tuned chemical processes occur in the blood to allow it to perform all of these functions and provide for the needs of the body. The tutorials in Chem 151 and 152 will describe several of these vital chemical processes, such as the release of iron in controlled amounts to the blood, the removal of waste products from the blood , and the regulation of the levels of CO2 and H+ to control the pH of the blood. In this tutorial, we will focus primarily on one of the most important functions of blood: the transport of oxygen from the lungs to the other cells of the body (e.g., muscle cells) that perform metabolic functions.
Metal-ion complexes consist of a metal ion that is bonded via coordinate-covalent bonds (Figure 1) to a small number of anions or neutral molecules called ligands. For example, the ammonia (NH3) ligand used in this experiment is a monodentate ligand; i.e., each monodentate ligand in a metal-ion complex occupies only one site in the coordination sphere of a metal ion. Some ligands have two or more electron-pair-donor atoms that can simultaneously coordinate to a metal ion and occupy two or more coordination sites; these ligands are called polydentate ligands. They are also known as chelating agents (from the Greek word meaning "claw") because they appear to grasp the metal ion between two or more electron-pair-donor atoms. One of the most important classes of chelating agents in nature is the porphyrins (Figure 1a). A porphyrin molecule can coordinate to a metal using its four nitrogen atoms as electron-pair donors, thus prophyrins are tetradentate ligands. The coordination number for a metal refers to the total number of occupied coordination sites around the central metal ion (i.e., the total number of metal-ligand bonds in the complex).
|Figure 1a (left). 2D representation of a porphyrin.
Figure 1b (right). A covalent bond forms when electrons are shared between atoms. A coordinate-covalent bond (represented by a green arrow) forms when both of the shared electrons come from the same atom, called the donor atom (blue). The top illustration shows a coordinate-covalent bond between a metal ion (e.g., Fe, shown in red) and a monodentate ligand (shown in light blue). The bottom illustration shows a metal ion with coordinate-covalent bonds to a bidentate ligand (shown in yellow).
This is a molecular model of hemoglobin with the subunits displayed in the ribbon representation which traces the backbone atoms of a protein. The four heme groups are displayed in the ball-and-stick representation and is used to represent the 3-D structure of the protein.
To understand the oxygen-binding properties of hemoglobin, we will focus briefly on the structure of the protein and the metal complexes embedded in it.
|Figure 3 |
This is a molecular model of the alpha-helix structure in a subunit of hemoglobin. The blue strands are shown in the ribbon representation to emphasize the helical structure. The green dotted lines show the hydrogen bonding between the -NH and -CO functional groups. Note: To view the molecule interactively, please use Jmol.
In the body, the iron in the heme is coordinated to the four nitrogen atoms of a porphyrin and also to a nitrogen atom of a histidine amino-acid residue in the hemoglobin protein (Figure 4). The sixth position (coordination site) around the iron of the heme is occupied by O2 when the hemoglobin protein is oxygenated.
On the left is a three-dimensional molecular model of heme coordinated to the histidine residue (a monodentate ligand) of the hemoglobin protein. On the right is a two-dimensional drawing of heme coordinated to the histidine residue, which is part of the hemoglobin protein. In this figure, the protein is deoxygenated; i.e., there is no oxygen molecule bound to the heme group.
Note: The coordinate-covalent bonds between the central iron atom and the nitrogens from the porphyrin are shown in gold; the coordinate-covalent bond between the central iron atom and the histidine residue is shown in green. In the three-dimensional model, the carbon atoms are gray, the iron atom is dark red, the nitrogen atoms are dark blue, and the oxygen atoms are light red. The rest of the hemoglobin protein is purple.
Note: To view the molecule interactively, please use Jmol.
Careful examination of Figure 4 demonstrates that the heme group is nonplanar when in its deoxygenated state; the iron atom is pulled out of the plane of the porphyrin toward the histidine residue to which it is attached. This nonplanar configuration is characteristic of the deoxygenated heme group and is commonly referred to as a "domed" shape. The valence electrons in the atoms surrounding the iron in the heme group and the valence electrons in the histidine residue form "clouds" of electron density. (Electron density refers to the probability of finding an electron in a region of space.) Because electrons repel one another, the regions occupied by the valence electrons in the heme group and in the histidine residue are pushed apart. Hence, the porphyrin adopts the domed (nonplanar) configuration in which the Fe is out of the plane of the porphyrin ring (Figure 5, left). However, when the heme group is in its oxygenated state, the porphyrin ring adopts a planar configuration in which the Fe lies in the plane of the porphyrin ring (Figure 5, right).
On the left is a schematic diagram showing representations of electron-density clouds of the deoxygenated heme group (pink) and the attached histidine residue (light blue).
On the right is a schematic diagram showing representations of electron-density clouds of the oxygenated heme group (pink), the attached histidine residue (light blue), and the attached oxygen molecule (gray).
The planar and nonplanar configurations of the heme group have important implications for the rest of the hemoglobin protein. When the iron atom moves into the porphyrin plane upon oxygenation, the histidine residue to which the iron atom is attached is drawn closer to the heme group. This movement of the histidine residue shifts the position of other amino acids that are near the histidine (Figure 6). When the amino acids in the protein are shifted by the oxygenation of one of the heme groups, the structure of the interfaces between the four subunits is altered. This causes the whole protein to change its shape. In the new shape, it is easier for the other three heme groups to become oxygenated. Thus, the binding of one molecule of O2 to hemoglobin enhances the ability of hemoglobin to bind more O2 molecules. This property of hemoglobin is known as cooperative binding.
This figure shows the heme group and the portion of the hemoglobin protein that is directly attached to the heme. As shown in the figure, the conformational change in the heme group upon oxygenation causes the entire hemoglobin protein to change its conformation as well. Please click on the pink button below to view a QuickTime movie showing how the amino acid residues near the heme group in hemoglobin shift as the heme group converts between the nonplanar (domed) and the planar conformation by binding and releasing a molecule of O2.
What causes this color change in the blood? We know that the shape of the heme group and the hemoglobin protein changes, depending on whether hemoglobin is oxygenated or deoxygenated. Therefore, these two conformations must have different light-absorbing properties. Recall that the color of a substance is the complementary color of what it absorbs. The oxygenated conformation of hemoglobin absorbs light in the blue-green range, and it reflects red light. This property accounts for the red appearance of oxygenated blood. On the other hand, the deoxygenated conformation of hemoglobin absorbs light in the orange range, and it reflects blue light. This property accounts for the bluish appearance of deoxygenated blood. We could use a spectrophotometer to examine a dilute solution of blood and determine the wavelength of light absorbed by each conformation.
|Figure 7 A schematic diagram of the flow of blood through the circulatory system
1. Blood rich in carbon dioxide is pumped from the heart into the lungs through the pulmonary arteries.
2. In the lungs, CO2 in the blood is exchanged for O2.
3. The oxygen-rich blood is carried back to the heart through the pulmonary veins.
4. This oxygen-rich blood is then pumped from the heart to the many tissues and organs of the body through the systemic arteries.
5. In the tissues, the arteries narrow to tiny capillaries. Here, O2 in the blood is exchanged for CO2.
6. The capillaries widen into the systemic veins, which carry the carbon-dioxide-rich blood back to the heart.
Note: The components of this diagram were not drawn to scale.
How do CO2 and H+ promote the release of O2 from hemoglobin? These species help form interactions between amino-acid residues at the interfaces of the four subunits in hemoglobin. These interactions are called salt bridges because they are formed between positively-charged and negatively-charged amino-acid residues on different subunits of the same protein (Figure 8). When salt bridges form, the subunits of hemoglobin are held in a position that "tugs on" the histidine that is attached to the heme iron. (Figure 5.) This favors the domed configuration, the deoxygenated form of hemoglobin.
On the left is a schematic diagram of the interface of two subunits of the deoxygenated hemoglobin protein. In the presence of CO2 and H+ (e.g., in the muscles), charged groups are formed on the amino-acid residues lining the subunit interface. These charged groups are held together by ionic interactions, forming salt bridges between the two subunits and stabilizing the deoxygenated form of hemoglobin. When blood passes through the alveolar capillaries of the lungs, CO2 and H+ are removed from the hemoglobin, and the oxygenated configuration is favored (right).
Note: The components of this diagram are not drawn to scale.
When the concentration of protons (H+) is low (pH 9), positive charges do not form on the residues at the subunit interfaces, so the salt bridges cannot form (right image in Figure 8). However, at pH 7, histidine residues at the subunit interfaces (not the histidine residues that bind the heme groups) can accept an additional proton (H+) and hence become positively charged (Equation 1). When salt bridges form by the interaction of these interfacial histidine residues and nearby negatively charged amino-acid residues, the deoxygenated hemoglobin structure is favored, and oxygen is released (left image in Figure 8).
The number of negatively charged residues in the salt bridges is increased in the presence of carbon dioxide. CO2 binds to the amino (-NH2) group of certain amino acid residues at the subunit interfaces to produce a negatively charged group (-NHCOO-) on the residue (Equation 2). This negatively charged group can form salt bridges with the positive charges on the protonated histidines described above. The H+ produced by Equation 2 can also be accepted by histidine residues at the subunit interfaces (via Equation 1).
Thus, the changing of the overall protein structure regulates hemoglobins biological function. This structure is altered by the binding or releasing of CO2 and H+ to the interfaces of the subunits in hemoglobin (Figure 8).
To view the molecules interactively, please use Jmol. To download the PDB files for viewing and rotating the molecules shown above, please right-click on the appropriate name below and choose 'Save link as' from the menu or click on the "interactive" button below each molecular-model figure in the text.
The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71199-502008 to Washington University.
Updated On: 2/26/2013