Myoglobin & Hemoglobin

Myoglobin & Hemoglobin
Evy Yulianti
Myoglobin- Key Properties

An O2 transport protein in muscle
A globular soluble protein, 151 residues (16 kDa)

8 a-helices (A,B,C,..H)- first protein crystal structure! Contains a heme prosthetic group
The Heme Prosthetic Group

The O2 carrier in Myoglobin and Hemoglobin
Protoporphyrin with Fe(II) Covalent attachment of Fe via His F8 side chain Additional stabilization via hydrophobic interaction Fe(II) state is active, Fe(III) *oxidized+
Fe(II) atom in heme binds O2
Binding of O2 to Heme
Binding of O2 to a free heme group is irreversible Enclosure in a protein allows reversible binding O2 has only limited solubility (1 X 10-4 M) in water Solubility problem overcome by binding to proteins Also increases diffusion Binding of O2 alters heme electronic structure Causes changes in heme electronic spectrum (Vis) Bright scarlet color of blood in arteries
Dark purple color of blood in veins
Carbon Monoxide Poisoning

Heme Fe(II) binds many other small molecules with structures similar to O2 including: CO, NO, H2S O2 is actually a fairly weak binder relative to these other molecules, particularly CO. [Essential for Biology] When exposed to CO, even at low concentrations, O2 transport proteins will be filled with CO limiting their vital O2 capacity.
Myoglobin O2 Binding Properties

Mb + O2 MbO2
Dissociation constant, Kd = [Mb] [O2] / [MbO2]
Quick Review [from enzymes]

If Kd (dissociation) is higher, binding is weaker
If binding curve is less steep, binding is weaker Half-saturation is the point where [O2] = Kd: Kd = [Mb] [O2] / [MbO2] = [Mb] Kd / [MbO2] Kd / Kd = [Mb] / [MbO2+ = 1 *Mb+ = *MbO2]

Mb + O2 MbO2, Kd = [Mb] [O2] / [MbO2]
To think about O2 transport, express binding in terms of the fraction of occupied binding sites, i.e. Fractional Saturation (YO2): YO2 = [MbO2] / ([Mb] + [MbO2]) Substituting with Kd, allows understanding of transport properties in terms of O2 availability: YO2 = [O2] / (Kd + [O2])

YO2 = [O2] / (Kd + [O2]) O2 is a gas: express [O2] as partial pressure (pO2) [Common to call pO2 oxygen tension] YO2 = pO2 / (Kd + pO2) As pO2 increases, binding increases Binding curve is hyperbolic, not linear (just like enzymes!!!) [Draw] Low pO2 little binding to protein High pO2 saturation of protein At half-saturation pO2 = Kd, field uses term P50 P50(myoglobin) = 2.8 torr / pO2 = 10-100 torr in-vivo!!
Hemoglobin- Key Properties

Ubiquitous O2 transport protein A globular soluble protein, 2X2 chains (164 kDa) a and b chains 44% identical All helical secondary structure (like myoglobin) abab quaternary structure a-subunit 141 residues b-subunit 146 residues Extensive contacts between subunits Mix of hydrophobic, H-bond, and ionic interactions a1b1 (a2b2)- 35 residues, a1b2 (a2b1)- 19 residues
Structure of Hemoglobin
a2
Heme
b1 b2
a1
Inter-subunit contacts
Hemoglobin O2 Binding Properties

Hb + nO2 Hb(O2)1 + Hb(O2)2 + Hb(O2)3 + Hb(O2)4
Kd = [Hb] [O2]n / [Hb(O2)1 + Hb(O2)2 + Hb(O2)3 + Hb(O2)4] *Hows that for complicated!?!?!+
To think about O2 transport, express binding in terms of the fraction of occupied binding sites, i.e. Fractional Saturation (YO2):
YO2 = [Hb(O2)1 + .+ / (*Hb+ + *Hb(O2)1 + .+)
Hemoglobin O2 Binding Curve

Binding curve is sigmoidal Artery: high pO2, loading of protein Vein: lower pO2, unloading from protein P50(hemoglobin) = 26 torr, adjusts as needed!!
*Drastic change in pO2 over physiological range*
Cooperativity in Binding O2
The sigmoidal shape is a consequence of the 4 subunits of hemoglobin "cooperating" in the binding of O2.
As pO2 increases and [O2] increases, increasing probability that at least 1 subunit has bound O2. Binding of O2 to a subunit INCREASES the probability that empty subunits will be able to bind an O2!! As pO2 increases even further, the probability that remaining binding sites will have O2 bound increases. Eventually, a plateau is reached: when most hemoglobins are filled there are few sites left to bind to, so not much increase, even if the pO2 is very high.
The Hill Equation

Simplification is required to solve binding equation: assume O2 binding cooperativity is infinite Binding curve can be expressed in terms of *O2]: YO2 = [O2]n / (Kd + [O2]n) O2 is a gas: substitute pO2 for [O2] and use Kd = P50: YO2 = (pO2)n / (P50 + (pO2))n This expression for the degree of saturation of Hb is known as the Hill Equation The quantity n is called the Hill Constant
Interpretation of the Hill Equation

YO2 = (pO2)n / (P50 + (pO2))n
n: the degree of cooperativity in ligand binding n = 1: non-cooperative n > 1: positive cooperativity implies binding of a ligand increases affinity for next ligand. n < 1: negative cooperativity implies binding of a ligand decreases affinity for next ligand.
Rearranging the Hill equation allows Hill Plots to be made to facilitate analysis of binding curves.
Binding of O2 to the Heme Changes the Whole Structure of Hemoglobin

R state T state
Shifts at the ab interfaces
b chains further apart
The T to R State Transition

Binding of O2 causes a series of shifts in all subunits Change in heme structure upon binding O2 Since His F8 is covalently attached, all of F helix shifts Reorganization of helix alters tertiary structure, which in turn alters the quaternary structure- 4 chains behave as a single cooperative structural unit Changes in packing of hydrophobic side chain Changes in pairing of charged side chains The change in conformation of Hemoglobin from the T to the R state increases O2 affinity at ALL sites
Allosteric Effectors
The R or T state can be stabilized by the binding of ligands other than O2. 1. H+. Lower pH favors the T state which causes Hb to release bound O2. This is known as the Bohr Effect. 2. CO2. Release of CO2 lowers pH via conversion to HCO3-: CO2 + H2O HCO3- + H+. Reinforces Bohr Effect 3. Bisphosphoglycerate (BPG). Regulation of activity via binding more strongly to T state, helps to release O2.
Increase in levels of BPG helps adaptation to high altitude- faster than making more hemoglobin. Also important in hypoxia diseases (e.g. anemia)
Towards a More Complete Picture

Model for disucssion
HEMOGLOBIN at the pH (~7.6) found in the lungs. HEMOGLOBIN at the pH (~7.2) found in peripheral tissues. MYOGLOBIN in muscle (a peripheral tissue).
Path of O2 Flow
1. O2 diffuses from the alveoli of the lungs into the capillaries of the bloodstream then into the red blood cells 2. In the red blood cells, O2 binds to hemoglobin. 3. In parallel, CO2 diffuses from blood into the alveoli. 4. The lower concentration of dissolved CO2 in the blood causes lower pH (~7.6) in lungs than in the peripheral tissues (~pH 7.2) where CO2 is being actively released.
A. High pO2 / high pH
Why O2 Transport Works

5. Red blood cells (containing O2-hemoglobin) carried to the peripheral tissues. B. pO2 decreases because O2 USED by the tissues. C. Blood plasma becomes more acidic (lower pH) because CO2 is released. The combination of lower pO2 and pH in the peripheral tissues causes a large decrease in O2 saturation. O2 is released by hemoglobin!!!!
Note: changes in pO2 and pH are small!
Why Myoglobin in Muscle?

Under resting conditions, O2 saturation is at point X on the green curve Small changes in pO2 and pH have very little effect on saturation During extremely vigorous exercise, heart pumps blood fast and breathing is rapid to increase the intake of O2 . Also, pH is lowered. Eventually, transport not fast enough to meet needs, i.e. pO2 lowered because O2 is used faster than it can be replenished. [Hemoglobin now no help!] Under extreme conditions, shift from point X to Y: saturation of the myoglobin is lowered = release of O2.
Defects from Hemoglobin Mutations

1. Weakened heme binding. 2. Disruption of secondary structure. 3. Disruption of quaternary structure. 4. Defective oxygen transfer. 5. Altered affinity for oxygen.
6. Oxidation of Fe(II) to Fe(III).

7. Aggregation in the T state (Hemoglobin S). Sickle cell anemia results from aggregation of Hb into insoluble fibers causing mishapen blood cells that cannot pass through capillaries and block blood flow to tissues.
Hemoglobin
A classic example of allostery Hemoglobin and myoglobin are oxygen transport and storage proteins Compare the oxygen binding curves for hemoglobin and myoglobin Myoglobin is monomeric; hemoglobin is tetrameric Mb: 153 aa, 17,200 MW Hb: two alphas of 141 residues, 2 betas of 146
Hemoglobin Function
Hb must bind oxygen in lungs and release it in
capillaries
When a first oxygen binds to Fe in heme of Hb, the heme Fe is drawn into the plane of the porphyrin ring This initiates a series of conformational changes that are transmitted to adjacent subunits
Hemoglobin Function
Hb must bind oxygen in lungs and release it in
capillaries
Adjacent subunits' affinity for oxygen increases This is called positive cooperativity
Myoglobin Structure

Mb is a monomeric heme protein Mb polypeptide "cradles" the heme group Fe in Mb is Fe2+ - ferrous iron - the form that binds oxygen Oxidation of Fe yields 3+ charge - ferric iron metmyoglobin does not bind oxygen Oxygen binds as the sixth ligand to Fe
See Figure 15.26 and discussion of CO binding
The Conformation Change

The secret of Mb and Hb! Oxygen binding changes the Mb conformation Without oxygen bound, Fe is out of heme plane Oxygen binding pulls the Fe into the heme plane Fe pulls its His F8 ligand along with it The F helix moves when oxygen binds Total movement of Fe is 0.029 nm - 0.29 A This change means little to Mb, but lots to Hb!
Binding of Oxygen by Hb
The Physiological Significance
Hb must be able to bind oxygen in the lungs Hb must be able to release oxygen in capillaries If Hb behaved like Mb, very little oxygen would be released in capillaries - see Figure 15.22! The sigmoid, cooperative oxygen binding curve of Hb makes this possible!
Oxygen Binding by Hb
A Quaternary Structure Change When deoxy-Hb crystals are exposed to oxygen, they shatter! Evidence of a structural change! One alpha-beta pair moves relative to the other by 15 degrees upon oxygen binding This massive change is induced by movement of Fe by 0.039 nm when oxygen binds See Figure 15.32
The Bohr Effect

Competition between oxygen and H+ Discovered by Christian Bohr Binding of protons diminishes oxygen binding Binding of oxygen diminishes proton binding Important physiological significance
See Figure 15.34
Bohr Effect II
Carbon dioxide diminishes oxygen binding Hydration of CO2 in tissues and extremities leads to proton production These protons are taken up by Hb as oxygen dissociates The reverse occurs in the lungs
2,3-Bisphosphoglycerate
An Allosteric Effector of Hemoglobin In the absence of 2,3-BPG, oxygen binding to Hb follows a rectangular hyperbola! The sigmoid binding curve is only observed in the presence of 2,3-BPG Since 2,3-BPG binds at a site distant from the Fe where oxygen binds, it is called an allosteric effector
2,3-BPG and Hb
The "inside" story...... Where does 2,3-BPG bind? "Inside"
in the central cavity
What is special about 2,3-BPG?

Negative charges interact with 2 Lys, 4 His, 2 Ntermini
Fetal Hb - lower affinity for 2,3-BPG, higher affinity for oxygen, so it can get oxygen from mother

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Myoglobin & Hemoglobin

Myoglobin- Key Properties

A globular soluble protein, 151 residues (16 kDa)

The Heme Prosthetic Group

Fe(II) atom in heme binds O2

Dark purple color of blood in veins

Carbon Monoxide Poisoning

Myoglobin O2 Binding Properties

Quick Review [from enzymes]

Myoglobin O2 Binding Properties

Myoglobin O2 Binding Properties

Hemoglobin- Key Properties

Hemoglobin O2 Binding Properties

Hemoglobin O2 Binding Curve

*Drastic change in pO2 over physiological range*

The Hill Equation

Interpretation of the Hill Equation

Binding of O2 to the Heme Changes the Whole Structure of Hemoglobin

Shifts at the ab interfaces

b chains further apart

The T to R State Transition

Towards a More Complete Picture

A. High pO2 / high pH

Why O2 Transport Works

Note: changes in pO2 and pH are small!

Why Myoglobin in Muscle?

Defects from Hemoglobin Mutations

6. Oxidation of Fe(II) to Fe(III).

See Figure 15.26 and discussion of CO binding

The Conformation Change

The Bohr Effect

See Figure 15.34

What is special about 2,3-BPG?

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Drastic change in pO2 over physiological range