Biochemistry 462a Hemoglobin Structure and Function Reading - Chapter 7 Practice problems - Chapter 7: 1-6; Proteins extra problems Myoglobin and Hemoglobin Oxygen is required for oxidative metabolism and energy production in most cells. Oxygen is used to oxidize dietary substrates to carbon dioxide, yielding energy. Vertebrates use hemoglobin (Hb) to transport O 2 to tissues from lungs (or gills) and to transport CO 2 from tissues to lungs (or gills). Mb is used in some tissues, notably muscle, as a storage reserve of O 2 and for intracellular transport. Mb is a monomeric protein, while Hb is a tetramer. However, each subunit of Hb has a tertiary structure very similar to the tertiary structure of Mb. In both Mb and Hb, the oxygen is carried bound to a iron-porphyrin complex called HEME. The IRON (blue in figure below) is in the ferric state (Fe +2 ) state. OXYGEN (red in figure below) binds reversibly to the Fe +2 without causing oxidation of the iron to Fe +3. Mb or Hb in which the iron has been oxidized chemically to Fe +3 does not bind oxygen; it binds water. Deoxy Heme Oxy Heme The heme group is buried within a hydrophobic pocket in the protein. The iron is coordinated to a histidine, THE PROXIMAL HISTIDINE. The hydrophobic character of heme-binding pocket is responsible for the lack of oxidation of Fe +2. 1
Oxygen Binding to Myoglobin The binding of O 2 to Mb follows a simple HYPERBOLIC saturation curve. The fractional saturation, θ, is defined by these equations. The partial pressure of O 2, P O2, is the fraction of the gas phase that is O 2 and P 50 is the partial pressure of O 2 required to give 50% saturation. Note that [Mb] = [free myoglobin] and [Mb- O 2 ] = [myoglobin that has bound O 2 ]. Oxygen Binding to Hemoglobin The O 2 saturation curve of Hb is SIGMOIDAL (S-shaped). Such a sigmoid saturation curve is diagnostic of a COOPERATIVE INTERACTION between different O2 binding sites on the protein. Initially, Hb is in a low affinity T-STATE. Binding of O 2 causes a conformational change in the Hb, which converts it to the high affinity R-STATE. Thus the sigmoid saturation curve is a composite of a low affinity and a high affinity curve. The cooperative binding of O 2 to Hb is an example of an ALLOSTERIC EFFECT, in which the binding of one ligand to a protein influences the affinities of the remaining unfilled binding sites. 2
The Hill Plot The Hill equation describes the binding of n molecules of O 2 to Hb by an equation similar to the one shown above:. A linear form is given by A plot according to this equation can be used to assess cooperativity in binding - the Hill Coefficient, n. For Mb, such a plot yields a straight line with n = 1.0 For Hb, the plot is not a straight line, but composed of three parts: At low and high po 2 the plot has a slope of 1. o These represent conditions where the first (T state) or last (R state) O 2 are being added/bound. o Under these conditions the Hb behaves as through it had a single binding site. At intermediate values of po 2 there is a slope > 1. This represents the region of cooperative binding. o The value of the slope po 2 = p50 gives n. o For Hb this is about 3.5. The maximal possible value for n would be 4, the number of subunits, but the Hill coefficient is always less than this maximal value. 3
The Significance of the Sigmoid Oxygen Binding Curve The significance of the sigmoid O 2 saturation curve of Hb can be appreciated from the graph to the right. While both Mb and Hb will be saturated with O 2 at the partial pressure of O 2 in the lungs, only hemoglobin will release significant amounts of O 2 at the partial pressure of O 2 present in the tissues. In fact, the O 2 released by Hb can then be taken up by Mb for O 2 storage in those tissues, such as muscle, that have significant amounts of Mb. Hemoglobin Structure and Cooperative O 2 Binding The differences in O 2 affinity between T-State (deoxy) and R-State (oxy) Hb can be understood in terms of the changes in quaternary structure that accompany the conversion of deoxy Hb to oxy Hb. o The shift from the deoxy to oxy conformation arises from the fact that in deoxy Hb the iron lies out of the plane of the heme ring, but when O 2 binding occurs, the iron moves into the plane of the heme ring. o Because the proximal His is bound to the Fe, it moves also, causing the F helix in which it is found to move. o This movement alters the structure of each subunit, which in turn alters the interaction between subunits at the interfaces. o This ultimately leads to the shift from the deoxy to the oxy conformation. o Because of the way in which the subunits are packed together, one subunit cannot shift from the deoxy to oxy conformation without causing the others to change also, even though the other subunits have not bound O 2. o Once the other subunits have shifted to the oxy conformation, the affinity of the unoccupied sites for O 2 increases because there are no steric blocks to O 2 binding, i.e., the conformational changes have already taken place. 4
The Bohr Effect In the tissues, the ph is lower due to the presence of CO 2.. The lowered ph causes Hb to lose O 2. This is known as the BOHR EFFECT and increases the delivery of O 2 to the tissues. The origin of the Bohr effect lies in the fact that deoxy Hb is a weaker acid than oxy Hb: A major contribution to the Bohr effect involves the C-terminal His of each β subunit. In the deoxy state, this His forms a salt bridge to Asp 94, if the His ring is protonated. The salt bridge stabilizes the protonated form of the His, increasing the pk a (weaker acid) in the deoxy state. The salt bridge does not form in the oxy state. Thus protonation of His-146 favors the deoxy conformation. Carbon Dioxide Transport The presence of CO 2 in the tissues alters the affinity of Hb for O 2 in two ways: 1. Through lowering the ph - the Bohr effect 2. Through the formation of carbamates by the N-terminal alpha-amino groups of Hb: The carbamate formation releases H +, which contributes to the Bohr effect, and the negative charge introduced allows formation of additional salt bridges, but only in the deoxy state. Thus, formation of the carbamate favors the deoxy state. 5
Bisphosphoglycerate The red blood cell contains high concentrations of 2,3-bisphosphoglycerate (2,3-BPG), which is an ALLOSTERIC EFFECTOR of Hb's affinity for O2. BPG binds strongly to the deoxy form of Hb, but only weakly to the oxy form. Thus, 2,3-BPG favors the deoxy conformation. The importance of 2,3-bisphosphoglycerate (BPG) as an allosteric regulator of hemoglobin's O 2 affinity High altitude adaptation Adaptation to high altitude is a complex physiological process that involves many slower events (e.g. increase in amount of Hb). One event that occurs within 24 hours is an increase in the content of 2,3-BPG in the erythrocyte. The effect of the increased concentration of BPG is to reduce the affinity of hemoglobin for O 2, which increases the efficiency of O 2 delivery to tissues. 6
Fetal respiration The fetus obtains O 2 by diffusion across the placenta. This diffusion is aided by the fact that fetal blood has a higher affinity for O 2 than does maternal blood. Fetal hemoglobin (HbF) is a tetramer that contains two α and two γ chains. The γ chains in HbF, which replace the β chains, have a low affinity for BPG. Thus, HbF has a higher O 2 affinity than does maternal HbA, which "delivers" O2 to HbF. The Physiological Transport of O 2 and CO 2 How do all these allosteric effects combine to allow Hb to carry O 2 from the lungs (gills) to the tissues and to carry CO 2 from the tissues to the lungs (gills)? o As shown below, Hb stripped of all its allosteric effectors has too high an affinity for O 2 to allow effective transport of O 2 to tissues. o However, the presence of both CO 2 in the tissues and BPG in the red blood cell create a situation in which O 2 is efficiently transported from lung to tissue. 7
We can summarize these events as follows: 1. In the lungs the partial pressure of O 2 is high, which overcomes any negative allosteric effects and causes complete oxygenation of Hb. 2. As Hb-O 2 enters the tissues the presence of CO 2 and a lowered ph combine to favor the deoxy conformation and release of O 2. 3. The presence of BPG aids the delivery of O 2 by favoring the deoxy conformation. 4. Deoxy Hb binds CO 2. 5. The deoxy Hb returns to the lungs where the ph is higher, the O 2 content higher and the CO 2 content is lower. All these factors favor reverse of carbamate formation (loss of bound CO2), deprotonation of His 146 and the formation of oxy Hb. Mutant Hemoglobins Several hundred mutant hemoglobins are known to exist. In most, a single amino acid replacement occurs in either the α or β chain of normal Hb A. Many of these changes cause no known effect, but several lead to pathologies associated with abnormal O 2 transport. First, let's look at HbS, sickle cell hemoglobin. In HbS there is a single amino acid replacement of a Val for Glu at position 6 of the β chain. This seemingly innocuous change places a hydrophobic sidechain on the surface of the protein. In the deoxy conformation the Val sidechain of a β chain in one HbS binds to the β chain of another HbS. This leads to polymer formation and precipitation of the deoxy HbS. This leads to red cell lysis and anemia. Mutant Hemoglobins - what can be learned from experiments of nature? Mutant hemoglobins provide unique opportunities to probe structure-function relations in a protein. There are nearly 500 know mutant hemoglobins and >95% represent single amino acid substitutions. About 5% of the population carries a variant hemoglobin. Some mutant hemoglobins cause serious illness. The structure of hemoglobin is so delicately balanced that small changes can render the mutant protein nonfunctional. The following images were generated using hemoglobin A (2hhb.pdb, C. Fronticelli, unpublished and 1 hho.pdb, B. Shaana, J. Mol. Biol. 17 31 (1984)), and the Swiss- PdbViewer to introduce the various mutations. Thus, the images are not derived from actual crystal structures but represent approximations to what those structures would probably look like. 8
Mutations that lead to loss of heme. The heme group can be dislodged by mutations in the heme-binding pocket. In Hb Hammersmith [Phe(42)βSer] the Phe in Hb A at position 42 in the β chain is converted to Ser. In Hb A the Phe blocks access of water to the heme pocket. But the smaller polar Ser allows water to enter the heme pocket. This causes the heme to be dislodged from the pocket. This produces a nonfunctional molecule. Mutations that disrupt the tertiary structure of a subunit. In the β chain there are two helices (Red and Green below) that pass so close together that there is only room for the H sidechain of Gly between them. In Hb Savannah [Gly(24)βVal] the presence of the Val sidechain forces the two helices apart. This leads to disruption of the tertiary structure of the β chain. In turn, this leads to an unstable hemoglobin molecule. This is Hb A with Gly This is Hb Savannah with Val 9
Mutations that stabilize Methemoglobin In order for hemoglobin to reversibly transport O 2 the iron must remain in the ferric (Fe +2 ) state. Oxidizing the iron to Fe +3 produces methemoglobin, which does not transport O 2. All known methemoglobins arise from mutations which provide a negatively charged oxygen atom as a ligand for the iron. The negatively charged oxygen stabilizes iron in the Fe +3 state. This is Hb Milwaukee [His(87)βGlu] in which the distal His, which normally protects the iron from becoming oxidized has been replaced by Glu, which stabilizes the Fe +3 state. Mutations that change the stability of the R State or the T State. Mutations at the α 1 -β 2 interface often interfere with the quaternary structure of hemoglobin. Such mutations can change the relative stabilities of hemoglobin's R and T states, thereby affecting the O 2 affinity of the mutant hemoglobin. Hb A has a p50 = 26 mm Hg. 10
In Hb Kansas [Asn(102)βThr] a critical hydrogen bond in the α 1 -β 2 interface between β 2 -Asn(102) and α 1 -Asp(94) that stabilizes the R state is lost. This means that hemoglobin remains in the T state. This mutant hemoglobin has a low O 2 affinity (p50 = 70 mm Hg). There is little cooperativity in O 2 binding (Hill coefficient = 1.3). The α 1 subnit is red and β 2 subunit is green. In Hb Yakima [Asp(99)βHis] a critical hydrogen bond in the α 1 -β 2 interface between β 2 -Asp(99) and α 1 - Tyr(42) that stabilizes the T state is lost. This means that hemoglobin remains in the R state. This mutant hemoglobin has a high O 2 affinity (p50 = 12 mm Hg). There is no cooperativity in O 2 binding (Hill coefficient = 1.0). The α 1 subnit is red and β 2 subunit is green. Crocodile Hemoglobin Crocodiles can remain under water for up to one hour and usually kill their prey by drowning them. How do the tissues of the crocodile get O 2 while submerged? Unlike deep diving whales, which use myoglobin to store O 2 in muscle, crocodiles rely on a unique allosteric effector of Hb to ensure delivery of O 2 to tissues. While submerged, metabolism produces CO 2, which is converted to HCO 3 - and it is the HCO 3 - that acts as the allosteric effector by binding to residues at the α 1 β 2 interface in deoxy Hb and stabilizing the deoxy Hb. This unique allosteric effect is only found in crocodile Hb. Crocodile Hb does not bind BPG due to mutations in the BPG-binding pocket. 11