BC - Ch 7 - Hemoglobin and Myoglobin

Firsts with Hemoglobin and Myoglobin (7)

1. First protein to be crystallized (1849)

2. First protein to have its mass accurately measured

3. First protein to be studied by ultracentrifugation

4. First protein to be associated iwth a physiological condition.

5. First protein to show that a point of mutation can cause problems

6. First proteins to have X-ray structures solved

7. Theories of cooperativity and control explain hemoglobin fxn

How many residues in Myoglobin?

Monomer is composed by 153 residues

Structure of Myoglobin

-8 α-helices (A-H)

-Contains a SINGLE Heme group tightly wedged betweeen EF helices

- A through D are pyrole groups

Structure of Hemoglobin

-α₂β₂dimer

-monomer is structureally similar to myglobin

-1 heme group in each subgroup

-gives RBC their red color

Function of Hemoglobin (4)

1. Transports oxygen from lungs ot tissues

2. O₂diffusion alone is too poor for transport in larger animals.

3. Solubility of O₂is low in plasma i.e. 10⁻⁴M

4. But bond to hemoglobin [O₂] = 10 ⁻² M or that of air

Two alternative O₂transporters are:

1. Hemocyanin: a Cu containing protein

2. Hemoerythricin: a non-heme containing protein

Myoglobin Function (5)

1. Myoglobin facilitates respiration in rapidly respiring muscle tissue.

2. The rate of O₂diffusion form capillaries to tissue is slow because of the solubiliyt of O.

3. Myoglobine increases solubility of O in muslces. (storage of O in muscles)

4. Myoglobin facilitates O diffusion (it binds the O₂)

5. O₂storage is also a fxn of myoglobin becauuse myoglobin concentrations are 10-fold greater in whales and seals than in land mammals.

General on the Heme Group

-Each subunity of hemoglobin or myblobin contains a heme.

-Binds one molecule of O.

-Heterocyclic porrphyrin derivative (protoporphyrin IX)

-The iron must be in the Fe(II) form or reduced form (ferrous oxidation) state.

Structure of Heme in the Hemoglobin

-2 hydrophobic side chains hold heme in place (Val E11 and Phe CD1)

-There are four pyrole groups surrounding it.

- The 5th ligand is.....

- The 6th ligand is O₂

-There is a hydrophobic pocket where the heme is wedged.

-The protein portion of the hemoglobin prevents oxidation that is irreversible.

-Proper confromation needs to happen for it to bind.

-The Fe is surrounded by methane bridges

The visible absorption spectra for hemoglobin

- Binding of oxygen rearranges the electronic distribution and alters the d-orbital nrg.

-This causes a difference in the absoprtion spectrum.

- The red color arises from the differences between the nrg levels of the d-orbitals around the ferrous atom

-Some orbitals contain electorns and others do not. There is an nrg difference between them - the size of the wavelength of the maximal absorbance band.

What color is deoxy Hb?

Bluish

What color is Oxy Hb?

Red

What is an easy method to determine the percent Oxygen boutd to hemoglobin?

Measure the absorption at 578 nm.

What happens when Fe(II) is oxidized to Fe(III)

It produces methemoglobin which is brown and coordinated with water in the sixth position.

-Dried blood and old meat have this brown color.

-Butchers use ascorbic acid to reduce methemoglobin to make the meat look fresh.

-There is an enzyme, methemoglobin reductase, that converts methemoglobin (Fe(III)) to regular (Fe(II)) hemoglobin.

Dissociateion Contant for myoglobin

Kd = ([MbO₂] [O₂])÷ [MbO₂]

O₂BInding to myoglobin

-At low pO₂very little O₂binds myoglobin.

- As pO₂increases, more binds

-The lower the valute of K, the tigher the binding

-i.e. YO₂=pO₂÷ (Kd +pO₂)₂

-The shape of the curve of the plot of YO₂b₂s pO₂ is a rectangular hyperbola

***What is P₅₀?***

The partial oxygen pressure when YO₂= 0.50

***What is the P₅₀ of Myoglobin?****

2.8 torr

When pO₂= K, how saturated is myoglobin

1/2 saturated.

1 torr =

= 1 mmHg = 0.133kPa

1 atm of pressure =

760 torr

What does the value of P₅₀ tell you about the O₂binding affinity of Mb?

Mb gives up litte O₂over normal physiological range of oxygen concentrations in the tissue

***What is the pressure in arterial blood?***

100 torr

***What is the pressure in venous blood?***

30 torr

What is the YO₂range for Mb

0.97 to 0.91

What is the P₅₀ value for Hemoglobin?

26 torr (10 x greater than Mb)

Hill plot

-Shows there is a decreas in affinity for last O in Hb

-Once O is bound, it will be easier to bind the next one

-Mb slope = 1 (no change in affinity for binding of O)

-Hb slope is sigmoidal ( by increasing concentration of O slope changes)

- 4th Hb is 100-fold affinity than first

_Since: P₅₀(1st O₂) = 30; P₅₀(4th O₂) = 0.3

Affinity for O for Hb

- Hb subunits independently compete of O₂for the first oxygen molecule to bind.

-When the YO₂is close to 1 i.e. 3 subunits are occupied by O₂binding to the last site is independent of the other sites

-Promotes conformation change, making it easeir to bind the next O₂

-When one molecule binds the rest bind and when one is released the rest are released.

In any binding system, a sigmoidal curve is diagnostic of...

a cooperative interxn between binding sites

YO₂of Hemoglobin in lungs

0.95 at 100 torr

YO₂of Hemoglobin in tissue

0.55 at 30 torr ( a ∆YO₂of 0.40)

Contrast MB O₂binding to Hb

Hb gies us easier than Mb and the binding is cooperative. For Mb, YO2 = 97 at 100 torr and 91 at 30 torr

Function of the globin (4)

1. Protoporphyrin binds oxygen to the 6th ligand of Fe(II) out of the plane of the heme. The 5th ligand is Histidine, F8 on the side across the geme plane.

2. His F8 binds the proximal side and the oxygen binds to the distal side.

3. The heme alone interxts with oxygen such that the Fe(II) becomes oxidized to Fe(III) and no longer binds oxygen.

4. The globin acts to modulate oxygen binding affinity and make reversible oxygen binding possible.

A heme dimer is formed which leads to the formation of Fe(III)

By introducing steric hindrance on one side of the heme plane intrxn can be prevented and oxygen bidng can occur

Oxygen binding to Hb triggers a conformational change from...

T (deoxyHb) to R (oxyHb)

The Bohr Effect

- Enhances O₂transport.

- The conformational changes in Hb that occur in O binding decr pKs of several groups

- I increase in pH (remove protons), it stimulates Hb to vind more O₂at lower O pressure

Higher pH and Hb

(lower [H+], basic) promotes tigher binding of oxygen to hemoglobin

Lower pH and Hb

(higher [H+], acidic) permits the eaiser release of oxygen from hemoglobin

Protons and Hb

Protons will intxt with side groups. Lower pH = easier release. Higher pH = difficult to release. See eq

****Equation on Bohr Effect***

Hb(O2 )n H x + O2 ⇔ Hb(O2)n +1 + xH +

Where n= 0,1,2,3 and x ≅0.6. A shig in the equilibrium will influence the amount of oxygen binding . Bohr protons

See graph

...

In R state Hb, what is absent?

Ion pairing is absent and pKs of group decreases.

How many protons does Hb release for each O₂it binds?

≈0.6 protons

What is the relation between pH and the P₅₀balues for oxygen binding?

As pH increases the P₅₀ values decreases, indicating the oxygen binding increases. The opposite effect ₀occurs when the pH decreases.

At 20 torr how much more oxygen is release at pH 7.2 than 7.4?

10% more

CO2 and O₂release

CO₂ is a biproduct of ATP making

Exercise = more CO₂ which lowers pH (incr [H+])

As oxygen is consumed, Co₂is released. Carbonic Anhydrase (enzyme) catalyzes this rxn in RBC. The H+ released from this rxn, induces further O₂release

CO₂ + H₂O ↔H+ + HCO₃⁻

How much CO₂is made for each O₂consumed?

0.8mol.

What happes to the H+ generated from the CO₂rxn?

The H+ generated from this rxn is take up by the Hb and causes it to release more oxygen. THis proton uptake facilitates the transport of Co₂ by stimulating bicarbonate formation.

What else does CO₂do?

It helps modulate O₂binding by binding reversible to the N-termini of the N-termini of the Hb domains by forming carbamates.

R-NH₂+ CO₂↔ R-NH-COO⁻ + H⁺ (Carbamates are formed with the interxn of CO₂with the Nterminal amino groups of Hb

Exercising muscles and lactic acid

Exercising muscles generate lactic acid so fast, lower pH from 7.4 to 7.2. At pO₂of 20 torr, Hb releases ≈ 10% more O₂at pH 7.2 than pH 7.4

BPG

D-2,3-bisphosphoglycerate binds to hemoblobin and DECREASES the oxygen affinity and keeps it in the deoxy form.

-It binds tightly to deoxyHb but weakly to oxyHb

-Binds 1:1 with a K = 1x10⁻⁵M to the doxyt

Tdeoxy HB binds more...

CO₂as carbamate than R form.

The P₅₀ value of stripped Hb increases from

12 to 22 torr by 4.7mM BPG

When is hemoglobin 95% saturated

At 100 torr (arterial blood)

When is Hb 55% saturated?

At 30 torr (venous blood)

How much O does the Hb release?

40%. In the absence of BPG little oxygen is release. Between BPG, Co₂, H⁺ and Cl⁻, all O₂binding is accounted for

BPG Levels and High-Altitude Adaptation

At 4500 m arterial pO₂is 55 torr and without the BPG adaptation only 30% of O₂is released.

-BPG restores the 37% release of O₂at higher elevations between arterial and venious blood.

THere is less saturation of Hb but the same amt of deliverty. When BPG incr, tincr affinity of Hb for O2.

Structure of Myoglobin

- 153 residues

-8 helices (A-H_

-Heme (porphyrin) with iron atom at the center

-O₂, CO, NO and H₂S can bind to the Fe(II) of the heme

-Heme corrdinates Fe by 4 N atoms

His F8 is the fifth ligand

-O₂or CO₂represent the sixth ligand

The heme group structure

-4 N atoms (pyrrole rings) of hem corrdinating hte FE(II)

-His F8(8th aa) is the fifth ligand

-O₂is 6th ligand

CO, NO and H₂S also bind

-CO binds a factor of 200 more times strongly than O₂to myoglobin

His E7 and binding

His E7 forms a H-bond with the bound O₂molecule.

The two hydrophobic residues above the heme group help hold the heme in place and help to prevent oxidation of the Fe.

Hemoglobin Structure

- Approximately spherical 64 x 55 x 50 A.

-Two-fold symmetry

α and β units are similar

-There is no D helix in the α-chain of hemoglobin

-Extensive interactions between unlike subunits

Extensive interactions between unlike subunits of Hb

α2-β2 or α1-β1 interface has 35 residues

-α1-β2 and α2-β1 have 19 residue contact

-No contact between α1-α2 or β1-β2. and they are separated by 20 A solvent (water) channel

-Contacts are predominately hydrophobic but there are some hydrogen-βbonds and salt links

2 Types of Hemoglobins

1. Deoxyhemoglobin (T)

2. Oxyhemoglobin (R)

Beta-monomers are related by 2-fold symmetry.

Space is smaller when O₂binds.

Binding and hemoglobin

Binding of the O on one heme is more difficult but its binding causes a shift in the α1-β2 contacts and moves the distal His E7 and Val E11 out of the oxygens path to the Fe on the other subunit.

This process increases the affinity of the heme toward oxygen.

The α1-β2 contacts have 2 stable positions.

These contacts which are joined by different but equivalent sets of H-bonds that act as a binary switch between the T (deoxy) and the R (oxy) states

Structure of the T-state

deoxyhemoglobin

Fe is 0.6 A out of heme plane

Structure of R state

Oxyhemoglobine

-Fe is in the heme plane

-Helix containing F8 shifts

-Change in quaternary structure

-C-terminal residues (Arg141α. His146β, val1α) change interactions and/or ionization state (Bohr effect.

Allosteric Interactions

The binding of the ligand at one site affects the binding of a ligand to another site.

Only the confromation and charge alters the affinity for the ligand

The molecular symmetry of the protein is conserved during the conformational change

Origin of Bohr Effect

1. The T→R transition causes the changes in the pKs of several groups

2. The N-terminal amino gorups are responsible for 20 to 30% of the Bohr effect.

3. His 146β accounts for about 40% of the Bohr effect salt bridged with Asp 94β.

4. This interaction is lost in the R state

Changes intrxn and wheterh they protoate or not

Decr ph→more protons bound, less affininty

Incr pH →less protons, more affinity

Networks of H-bonds and ion pairs in T-state

The T state is shown above.

- T→R transition bauses breakage of terminal interactions and changes in ionization states of His 146β and Val1α (part of Bohr effect)

Is subunits are so tightly coupled that large tertiary structural changes winin one subunit cannot occur without quaternary changes in the entire tetrameric protine.

Roles of Myoglobin and Hemoglobin

0.6H⁺released for each O₂binding

CO₂+ H₂O→H⁺ + HCO₃⁻, catalyzed by carbonic anhydrase (greatly accelerates rxn) - main mode of of elimination of CO₂

R-NH₂+ CO₂-R-NH-COO⁻(carbamate) + H⁺, formed at N-termini helping to stabilize the T-state (deoxy

Binding of BOG to deoxyhemoglobin

BPG binds specifically to the deoxy state and stabilizes it in the T-state.

In the R-state, the central cavity is too narrow for BPG to fit or bind.

The presence of BPG decreases Hb's O affinity by keeping it in the deoxy conformations.

If it wasn't there, O₂would never be released because of O affinity

General on Hemoglobin mutants

There are about 500 variants of hemoglobin

95% are single aa substitutions

5% of the world's populations carries a differnt sequence from the normal.

Types of changes in Hemoglobin Mutants (4)

1. Changes in surface charge

2. Changes in internally located residues

3. Changes in stabilizing Methemoglobin (oxidized Fe(III))

4. Changes in the α1-β2 contact

Hemolytic anemia

Lysing of the cell compromises O₂delivery

Sickled erythrocytes

1. Lower number of RBC (anemia)

2. Single AA change in Glu. Modified to Glu6Val

3. Fibers make difficult for sickle shaped cells to flow through the capillaries

4. Can completely block blood flow and leads to hemolytic anemia

5. 10% African American

6. Greatest danger when erythrocytes pass through capillaries wherer deoxygenation occurs.

Hemoglobin S fbers

Hemoglobin S (sickle) forms fibers, causing deformation of the erthrocytes

Caused by a single Glu6Val (loses negative charge)

This mutation allows formation of fibers by stabilizing contacts - Val inserts into a hydrophobic pocket.

Promotes interxn with each other and congregate together.

Glu6Val mutation on β1 allows formation of S-fibers.

Val6 inserts into a hydrophobic pocket on β1 so then pocket is abstent Glu and absent oxyhemoglobin

What is myoglobins major physiological role?

To facilitate oxygen diffusion in the muscle. It increases the effective solubiliyt of O2 in the muscle cells, acting as a kind of molecular bucket brigade to boost the O2 diffusion rates

Conditions of high exertion have a particularly high demand for O. Aquatic mammals there is a 10-fold higher concentraiton thatn in terrestrial animals

General structure of Myoglobin

Single polypeptaide containing a heme group, which consists of a porphyrin ring whose coordingated F(II) atom binds molecurar oxygen. The protein prevens oxidation of the heme iron to Fe(III) which does not bind oxygen

Metmyoglobin

Oxidized myoglobin

Simple equilibrium equation descibing O₂binding to myoglobin (Mb)

Mb + O₂↔ MbO₂

The fractional saturation of myoglobin

Yo₂= pO₂÷ (K + pO₂) where PO₂is the partial pressure of oxygen and K is the dissociation constant.

A plot of Yo₂ versis pO₂ is a ...

hyperbolic curve

K =

p₅₀, the partial pressure of O₂at which 50% myoblobin has bound oxygen. p₅₀ = 2.8 torr, which is much lower than the pO₂ in venous blood (30torr), so that myoblobin is nearly saturated with oxygen under physiological conditons.

Physiological purpose of Hemoglobin?

Binds oxygen in the lungs (pO₂= 100 torr) and releases it in the capillaries (pO₂= 30 torr). The efficiency of oxygen transport is greater than expected if oxygen binding were hyperbolic, as in myoglobind.

General Strucure of hemoglobin

α2β2 tetramer, each of whose subunits contain a heme gorup, binds O₂cooperatively and thus has a sigmoidal oxygen binding curve. Deoxyglboin is bluish (the color of venous blood) whereas oxyhemoglovind is bright red (the color of arterial blood.

p₅₀ of hemoglobin

Approximately 26 torr, which is nearly 10 times greater than that of myoglobin. Because Hb exhibits a sigmoidal oxygen-binding curve, it realsease a much greater fraction of its bound O₂in passing form the tlungs to the tissues than if it had a hyperbolic binding curve with the same p50.

The Hill equation

Describes the cooperative nature of oxygen binding, and the hill constant , describes the degree of cooperativity. The Hill constant, which is not necessarily and integer, is obtained experimentally,. The binding of O2 to Hb is said to be cooperative because the binding of O2 to one subuinit increases the O2 affinity of the other subunits. THe fourth oxygen to bind Hb does os with a 100-fold greater affinity than the first

Other oxygen transport protiens

Leghemeoglobins from leuminous plants

Chlorocruorins from some annelids

Hemerythrin and Hemocyanic from invertebrates (unique b/c don't contain heme groups)

T↔R state

Hb has only two stable conformation states, the T state (deoxyHb) and R state (oxyHb). Oxygen binding causesgreaterthe T state to shift to the R state, which has greater affinity for O2. The T to R shift is triggered by O binding to the heme iron, which pulls the heme iron atom into the heme plane. This movement is transmitted to the F helix thorugh His F8, which ligands the iron atom. Conformational changes in one subunit are tansmitted across the α1-β2 and α2-β1 interfaces. Due to the conformational constraints at these interfaces, the conformational shift of one subunit must be accompanied by the conformational shift of all subunits, thereby increasing the O afinity of the unoccupied subunits

Bohr effect

Decreases in pH promote the release of O from Hb. It is driven by dissoved carbon dioxide which forms bicarbonate ion and H ion. The H ion protonates Hb, thereby stabilizing its T (deoxy) state. In the lungs, the rxn is reversed: O binding causes a switch to the R (oxy) state. The released H ions hshift the equilibrium between bicarbonate and carbon dioxide, thereby forming carbon dioxide for expulsion form the lungs. Due to the Bohr effect, the low pH in highly active muscles causes the amount of O delivered by Hb to increase by nearly 10%. Carbon dioxidealso binds preferetially to the N-terminal amino groups of T-state Hb as carbamates and hence is released by R-state Hb. This accounts for about half of the carbon dioxide release from the from the blood in the lungs

BPG

Hb stripped of 2,3 -biphosphoglycerate (BPG) binds oxygen more tightly than Hb in the blood. BPG binds to the T state but no the R state, thereby decreasing Hb's oxygen affinit. This allows nearly 40% of the oxygen to be unloaded in venous blood. Fetal Hb does not bind BPG as tightly and therfore has a higher affinity for O than adult Hb

Humans use 2 mechanisms to adapt to lower oxygen partial pressure at higher altitudes:

1. Erytrocyte BPG concentration increases to promote oxygen delivery to tissues, and

2. The Hb concentations rises.

Other mammels that live at high altitue=de have isoforms of Hb with increased affiniaty for O

Allosteric proteins

Are oligomers with multiple ligand-binding sites, in which ligand binding at one site alters the protein's binding affinity for ligand at another site. Tow modes for cooperativity have been proposed:

1. Symmetry model

2. the Sequential model

Symmetry Model

Proposes that all the subunits in the protein exist in either the T state or the R state, and the ligand binding to one subunit facors theconversiton of all subunits to the R state.

Sequential Model

Proposes that ligand binding induces a conformational change in one subunit (to the Rs state) which cause progressive changes in conformation in adjacent subutes. C urrent sutdies suggest that Hb shows features of both models, neigher of which can fully explain the complexity of protein dynamics upon ligand binding.

Hemoglobin variants that have demonstated clinical symptoms are most commonly correlated with (4)

1. decreased ability of globins to bind heme

2. decreased cooperativiyt in which the protein is "locked" in either the T- or R- state

3. Increased porpensity for oxidation of the ferrous iron to the ferric state

4. decreased protein stability in protein degradation

Hb S

Has a substitution of valine for glutamic acid in the β subunit. In the deoxy state Hb S aggregates, causing erythrocytes to sickle and block the capillaries. Individuals who are homozygous for the gene specifying Hb S have sickle cell anemia, a debilitating and often fatal dz.