Hemoglobin Structure and Function: Oxygen Binding and Hemoglobinopathies

Hemoglobin Structure and Function

UAG. Universidad Autónoma de Guadalajara SCHOOL OF MEDICINE WE MAKE DOCTORSHEMOGLOBIN STRUCTURE AND FUNCTION Physiology Department

Objectives

• Describe the role of hemoglobin in oxygen transport.
• Identify the structure of hemoglobin and describe its constitutional changes during O2 take-up.
• Explain the hill coefficient and its role in the oxygen dissociation curve. Contrast the oxygen dissociation curve of Myoglobin and Hemoglobin.
• Explain the cooperativity effect during O2 binding
• Explain the differences between deoxy- and oxyhemoglobin.
• Describe the allosteric modulation of the 02 affinity of hemoglobin.
• Explain the relationship between PO2 and Hb saturation (%) with regards to the oxyhemoglobin dissociation curve.
• Explain the clinical significance of each portion of the oxyhemoglobin dissociation curve
• Describe the physiological factor that shift the oxyhemoglobin dissociation curve.
• Define the Bohr effect.
• Identify examples of hemoglobinopathies and contrast their biochemical changes.

UAG SCHOOL OF MEDICINE 3

Hemoglobin: An Oxygen Transport Protein

Hb is the principal O 2 -transporting protein in human blood; it is localized exclusively in erythrocytes

• Oxygen travels through blood in two forms:
• Dissolved (a small amount - PaO2) and bound to Hgb.
• Bound O2 to Hgb > Oxyhemoglobin (primary transport mechanism of O2).
• O2-carrying capacity of blood > 1 65 times by its ability to bind to Hgb.

Red blood cell Oxygen from lungs Oxygen released to tissue cells Hemoglobin molecules Oxygen bonded with hemoglobin molecules UAG SCHOOL OF MEDICINE

Hemoglobin: Structure and Subunits

Hb is the principal O 2 -transporting protein in human blood; it is localized exclusively in erythrocytes

• HbA -> Tetrahedral array
• 2 identical a-globin
• 2 identical ß-globin
• Each globin subunit contacts the other 3
• Multiple non-covalent interactions -> Pairs of dissimilar subunits (a-ß)
• Less hydrophobic interactions > a1-a2 or 31 - 32

Heterodimer interface B2 1 02 Heme iron B1 UAG SCHOOL OF MEDICINE Universidad Autónoma de Guadalajara A.C.@ 2022 5

Hemoglobin: Conformational Changes with Oxygen

Hb is the principal O 2 -transporting protein in human blood; it is localized exclusively in erythrocytes

• The number and nature of contacts change in the presence or absence of O2.
• Hb -> dimer of heterodimers (aß) 2

Weak ionic and hydrogen bonds occur between aß dimer pairs in the deoxygenated state. Strong interactions, primarily hydrophobic, between a and B chains form stable aß dimers. O2 O2 ... .... aß dimer 1 4 02 .... .... .... ... ... .... aß dimer 2 ... .... O2 O2 "T," or taut, structure of deoxyhemoglobin "R," or relaxed, structure of oxyhemoglobin Copyright & 2014 Waltes Klunes Health | Lippincott Williams & Wilkins UAG SCHOOL OF MEDICINE Some ionic and hydrogen bonds between aß dimers are broken in the oxygenated state. .... aß dimer 1 ... .... aß dimer 2 4 O2

Interactions of Hemoglobin with Oxygen

Hb binds oxygen cooperatively, with a Hill coefficient of ~2.7

• Hb is a gas delivery vehicle
• Binds 02 > entering the alveoli
• Releases 02 -> Through tissue capillaries
• Dual function by cooperative interactions among globin subunits.
• Biding of O2-Iron >Conformational changes
• New orientation of atoms (heme structure)

. Histidine and helix F shift positions > Structural realignment (globin subunit) . 12-15° rotation and 0.10 nm displacement of α · Structural changes: · Positive cooperativity 1 Increase biding affinity • · Negative cooperativity · Į Decrease biding affinity A B PheCD1 HisF8 HisE7 Fe2+ 2 F helix V E helix Proximal side Distal side UAG SCHOOL OF MEDICINE 7

Hemoglobin Oxygen Binding and Cooperativity

Hb can bind up to four molecules of O 2 in a cooperative manner

• Multiple ligand-binding sites and structural changes -> 02 binding
• Hill coefficient > Cooperativity among ligand-binding sites
• Adult Hb -> n = 2.7
• Hb > O2 affinity
• P50 of 27+ 2 mmHg
• Myoglobin ( P 50 = 4 mmHg)
• Decrease/Absence of cooperativity > Hb mutants -> Lost functional subunit-subunit contacts

100 Myoglobin 80 O2 saturation (%) Hemoglobin 4 hemes found in RBC O2/CO2 transport 60 40 Myoglobin P50 P50 20 1 0 30 60 90 Po2 (mm Hg) 1 heme found in muscle O2 storage UAG SCHOOL OF MEDICINE Universidad Autónoma de Guadalajara A.C.@ 2022 8 Hemoglobin

Hemoglobin Conformations and Oxygen Affinity

Hemoglobin subunits may assume two different conformations that differ in O 2 affinity

Affected by O2 binding (R-state) No oxygen (T- state) Fully oxygen (R- state) High O2 affinity Low O2 affinity Entering lungs K ×1| K Kı KA Exiting lungs Oxygen bound

• Binding of the first O2 favors the rest - cooperativity
• Cooperativity increases as it passes through the lungs
• Transition between states > breaking of noncovalent bonds

UAG SCHOOL OF MEDICINE Universidad Autónoma de Guadalajara A.C.@ 2022 9

Deoxy T state

A Deoxy T state Tense Low affinity B2 0 Asn 102 HON Asp94 0.57 0 O 0.30 HN 01 SCHOOL OF MEDICINE

Oxy R state

B Oxy R state Relaxed High affinity Asn 102 B2 0 Asp94 0 H2N 0.28 0 0.38\ 1 1 HN el 990

Allosteric Modulation of Hemoglobin Oxygen Affinity

Allosteric proteins and effectors

• Hb -> allosteric protein
• O2 affinity regulated by small molecules
• Allosteric effectors > long-range conformational effects > alter ligand-/substrate-binding affinity of the protein
• Allosteric proteins = multiple-subunit proteins
• H +, CO 2, and 2,3-bisphosphoglycerate (2,3- BPG).
• Homotropic > oxygen affects its own binding at other sites
• Heterotropic -> effector (i.e., Ph on O2 binding) affects binding of oxygen
• Leading to horizontal shifts in the O2 binding curves

UAG SCHOOL OF MEDICINE Universidad Autónoma de Guadalajara A.C.@ 2022 Peripheral tissue Lung 1.00 A B 0.75 A-B (R)-(T) 0.50 - + [H+] + [CO2] 1 [2,3-BPG] { 0.25 0.0 0 201 40 60 80 100 120 P50 values pO2 (mmHg) O2 saturation (Y)100

Oxy-hemoglobin Dissociation Curve

20 Left shift 90 18 80 16 % Hgb saturation 70 14 Right shift 60 12 P50 50 10 40 8 30 - 6 20 - 4 10 F 2 0 - 0 10 20 30 40 50 60 70 80 90 100 27 Po2 (mm Hg) Oxy-hemoglobin Dissociation Curve

• In the alveoli, the majority of O2 in plasma quickly diffuses into red blood cells and chemically binds to Hgb.
• The binding process is reversible, so that Hgb quickly gives up its O2 to tissue through passive diffusion.
• The oxyhemoglobin dissociation curve illustrates the relationship between PO2 in blood and the number of O2 molecules bound to Hgb.

UAG SCHOOL OF MEDICINE Q2 content mL/100 mL (vol %)

Clinical Significance of the Oxyhemoglobin Dissociation Curve

S shape: dependence of Hgb saturation on PO2, especially at partial pressures lower than 60 mm Hg. The clinical significance of the steep portion (<60 mm Hg) of the curve is that a large amount of O2 is released from Hgb with only a small change in PO2, which facilitates the release and diffusion of O2 into tissue. 20 100 Left shift 90 - 18 80 - 16 % Hgb saturation 70 - 14 Right shift 60 12 P 50 50 - 10 - 8 30 - 6 20 F 4 10 - - 2 0 0 10 20 30 40 50 60 70 80 90 100 27 PO2 (mm Hg) The point on the curve at which Hgb is 50% saturated with O2 is called the P50, and it is 27 mm Hg in normal adults. The clinical significance of the flat portion of the curve (>60 mm Hg) is that a drop in PO2 over a wide range of partial pressures (100 to 60 mm Hg) has a minimal effect on Hgb saturation, which remains at 90% to 100%, a level sufficient for normal O2 transport and delivery. UAG SCHOOL OF MEDICINE Q2 content mL/100 mL (vol %) 40

Physiological Factors Shifting the Oxyhemoglobin Dissociation Curve

O2

• Can shift either to the right or to the left.
• Right when the affinity of Hgb for O2 decreases, which enhances O2 dissociation and causes the P50 to increase.
• Left when the affinity of Hgb for O2 increases, which causes the P50 to decrease.

UAG SCHOOL OF MEDICINE Decreased Pso (increased affinity) 100 Temperature Hemoglobin saturation (%) + Pco2 80 + 2,3-DPG 1 pH 60 Increased P50 (decreased affinity) 1 Temperature 40 T 1 Pco2 1 2,3-DPG 20 Į PH 0 - 0 20 40 60 80 100 Oxygen partial pressure (mm Hg)

Factors Affecting Oxygen Binding

Decreased P50 (increased affinity) 100 Į Temperature Hemoglobin saturation (%) 1 PCO2 80 + 2,3-DPG Î pH 60 Increased P50 (decreased affinity) 1 Temperature 40 1 PCO2 1 2,3-DPG 20 Į pH 0 0 20 40 60 80 100 Oxygen partial pressure (mm Hg) UAG SCHOOL OF MEDICINE Factors Affecting O2 Binding

• pH: When the pH of the blood is below 7.35, the affinity of Hb for oxygen is reduced and is mostly associated with increases in CO2 in the plasma and RBC.
• CO2: an increased CO2 concentration leads to reduction in pH and consequently in the affinity of the oxygen for hemoglobin.
• The effect of pH and CO2 on the oxygen/hemoglobin affinity is known as the Bohr effect.

The Bohr Effect

(pH) Increased CO2 production by tissue and its release into blood results in the generation of hydrogen ions (H+) and a decrease in pH. This shifts the dissociation curve to the right, aiding in the release of O2 from Hgb, this is due to the decrease in pH and to a direct effect of CO2 on Hgb. Conversely, as blood passes through the lungs, CO2 is exhaled, increasing pH, and shifting oxyhemoglobin dissociation curve to the left. Bohr effect curves 100 80 Hb, % 02 saturation 60 High CO2 (low pH) 40 Normal CO2 (normal pH) Low CO2 (high pH) 20 Body tissues Lungs alveoli 0 20 40 60 80 100 Oxygen pressure, mm Hg UAG SCHOOL OF MEDICINE In other words, oxygen is released more rapidly by hemoglobin in tissues with an already high CO2 content.

Additional Factors Affecting Oxygen Binding

Factors Affecting O2 Binding

• 2,3-Bisphosphoglycerate (2,3- BPG): binds to deoxyhemoglobin. Maintains T form by stabilizing the ionic bonds of Hb. Decreases Hb's affinity to O2.
• Temperature: At higher temperature less affinity of Hb for O2 . Actively metabolizing tissues are warmer so an increase in O2 unloading occurs in these more active tissues.

UAG SCHOOL OF MEDICINE 2,3-BPG = 0 (Hemoglobin stripped of 2,3-BPG) S N % Saturation with O2 (Y) 100 г 2,3-BPG = 5 mmol/L (Normal blood) 2,3-BPG = 8 mmol/L (Blood from individual adapted to high altitudes) 0 0 40 80 120 Partial pressure of oxygen (mm Hg)

Carbon Monoxide and Hemoglobin

Factors Affecting O2 Binding

• Carbon Monoxide: CO binds to the heme group of the Hgb molecule at the same site as O2 and forms carboxyhemoglobin (HgbCO).
• The affinity of CO for Hgb is about 200 times greater than it is for O2.
• Small amounts of CO can greatly influence the binding of O2 to Hgb.
• In the presence of CO, the affinity of Hgb for O2 is enhanced causing the dissociation curve to shift to the left.
• As the PCO of blood approaches 1 mm Hg, all the Hgb binding sites are occupied by CO, and Hgb is unable to bind to O2 (levels above 5% to 7% are considered hazardous).

UAG SCHOOL OF MEDICINE CO + hemoglobin 100 Hemoglobin saturation (%) 80 60 O2 + hemoglobin 40 - 20 0 0 20 40 60 80 100 PO2 (mm Hg)