Oxygen Binding Proteins

Objectives:

• Hemoproteins carring O2

• Myoglobin function and structure

• Hemoglobin function and structure and forms

• O2 binding to myoglobin and hemoglobin

• O2 dissociation curve

• Allosteric effectors

Why do we need oxygen carriers ?i. Cannot carry enough in blood to meet metabolic demand

ii. Oxygen is very reactive – oxidizes

iii. Oxygen cannot diffuse easily (we have thick skin)

Properties of a good oxygen carrier

i. Binds oxygen at a high [O2]

ii. Doesn’t oxidize cellular components

iii. Gives up oxygen on demand

• Oxygen binding proteins (myoglobin and hemoglobin) are hemoproteinsOxygen binding proteins (myoglobin and hemoglobin) are hemoproteins

• HemoproteinsHemoproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group.

• Heme is a complex of protoporphyrin protoporphyrin IX & ferrousferrous iron (Fe2+) .

• Fe2+ is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring.

• It can form two additional bonds, one on each side of the planar porphyrin ring.

- one of these positions is coordinated to a histidine of globin

- the other position is available to bind oxygen

Oxygen Binding Proteins are Hemoproteins

The heme group: Fe2+ porphyrin complex with bound O2

OXYGEN

HISTIDINE

PORPHYRIN IX RING

FERROUS ION

• Location: heart & skeletal muscles giving them red color

• Function: a reservoir for oxygen for muscles. an oxygen carrier that increases

the rate of transport of oxygen within the muscle cell.

• Structure: Myoglobin is composed of a single polypeptide chain that is

structurally similar to the individual subunit polypeptide chain of hemoglobin.

The polypeptide chain is folded into 8 stretches labeled A to H

1- Myoglobin

Globin Protein Globin Protein (Polypeptide Chain)(Polypeptide Chain)

The interior almost entirely of nonpolar

amino acids.

Polar amino acids are located almost

exclusively on the surface of the

molecule.

Heme groupHeme group

is located in a pocket in the

molecule between helix E and

helix F which is lined by nonpolar

amino acids with exceptions

of two histidine amino acids.

Myoglobin cont.

• The first is called the proximal histidine (F8), binds directly to the iron of heme.

• The second is called the distal histidine (E7), does not directly interact with the heme group, but helps stabilize the binding of oxygen to the ferrous iron.

• The protein, or globin, portion of myoglobin prevents the oxidation of iron of heme.

Myoglobin cont.

Myoglobin as a diagnostic tool:Myoglobin as a diagnostic tool:

•Myoglobin is used as a marker for acute myocardial infarction

•Advantage: It is elevated in blood of patients with myocardial infarction than other markers as CK-MB or troponin. So, it can diagnose myocardial infarction attack at an early stage.

•Disadvantage: myoglobin has a reduced specificity for diagnosing myocardial infarction.

Myoglobin cont.

• Location: exclusively in red blood cells

• Function: main function is to transport oxygen, H+ & CO2 and act as RBCs buffer

• Structure: Hemoglobin is composed of four polypeptide chains - two α chains & two β

chains – arranged into 2 dimers held together by noncovalent interactions. • Each subunit has 8 stretches of α-helical structure & a heme-binding pocket

(as for myoglobin)

The oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors

2 -Adult Hemoglobin HbA1

• The hemoglobin tetramer can be envisioned as being composed of two

identical dimers, (αβ)1 & (αβ)2 (1 & 2 are numbers)

• The two polypeptide chains within each dimer are held tightly together, primarily by hydrophobic interactions

• In contrast, the two dimers are able to move with respect to each other, being held together primarily by polar bonds (ionic and hydrogen).

• The weaker interactions between these mobile dimers result in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin

Quaternary structure of hemoglobin

Structural changes resulting from oxygenation & deoxygenation of

hemoglobin

RELAXED Structure

R

TAUTStructure

T

Quaternary structure of hemoglobin cont.

T form: The two αβ dimers interact through a network of ionic bonds that constrain the movement of the polypeptide chains. The T form is the low-oxygen-affinity form of hemoglobin.

R form: The binding of oxygen to hemoglobin causes the rupture of some of the ionic bonds between the αβ dimers. This leads to a structure called the “R,” or relaxed form, in which the polypeptide chains have more freedom of movement The R form is the high- oxygen-affinity form of hemoglobin.

Quaternary structure of hemoglobin cont.

• Myoglobin can reversibly bind only one molecule of oxygen (O2), as it contains only one heme group.

• Hemoglobin can bind four oxygen molecules (one at each of its four heme groups) cooperatively as 1st O2 bind at one heme increases the oxygen affinity of the remaining heme groups in the same hemoglobin molecule.

• The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100% (all sites are full

Binding of oxygen to myoglobin & hemoglobin

The oxygen dissociation curve is

a plot of saturation of binding sites with O2 in hemoglobin or myoglobin (Y) measured at

different partial pressures of oxygen (pO2)----------------------------------------------------------

Myoglobin has a higher oxygen affinity at any pO2 value than

hemoglobin as:

In myoglobin:

pO2 needed to achieve 50% saturation of

binding sites is ~ 1 mm Hg

In hemoglobin:

~ 26 mmHg

Oxygen dissociation curve

The ability of hemoglobin to The ability of hemoglobin to reversiblyreversibly bind oxygen is affected by: bind oxygen is affected by:

• pO2 • pH• pCO2• 2,3-bisphosphoglycerate (2,3 BPG) • CO

These are collectively called allosteric (“other site”) effectors, because their interaction at one site on the hemoglobin molecule affects the binding of oxygen to

heme groups at other locations on the molecule.

N.B.: The binding of oxygen to myoglobin is not influencedby allosteric effectors.

Allosteric effectors

Loading & unloading of oxygen depend on pO2 (oxygen concentration

1- pO2 in alveoli of lungs (i.e. concentration of O2) is high

So, affinity of Hb to O2 is increased leading to saturation of hemoglobin with O2 (loading of oxygen)

2- pO2 in peripheral tissues is low

So, affinity of Hb to oxygen is decreased leading to release of oxygen to peripheral tissues (unloading of oxygen)

AT THE SAME TIME:

Myoglobin is designed to bind oxygen released from hemoglobin at low pO2

found in muscles (as myoglobin requires very low pO2 to be fully saturated

with oxygen)

1- pO2 (Oxygen Concentration)

• Bohr effect is the change of oxygen binding in hemoglobin due to Bohr effect is the change of oxygen binding in hemoglobin due to hydrogen ions (H+ or protons) and CO2hydrogen ions (H+ or protons) and CO2

• In peripheral tissuesIn peripheral tissues, , H+H+ & & CO2CO2 are increased, which results to increased are increased, which results to increased release of oxygen from hemoglobin.release of oxygen from hemoglobin.

1- 1- H+H+ released from metabolism of peripheral tissues released from metabolism of peripheral tissues

2- CO2 resulting from cellular metabolism is converted by carbonic anhydrase to carbonic acid which is converted to bicarbonate & H+

• In both cases, H+ increases the ionic bonds favoring of T form of Hb

O2 release to the tissues

• Hb O2 (OxyHb, R form) + H+ ↔ HbH (deoxy Hb, T form) + O2 So, increase in H+ shift equilibrium to right

2- Bohr effect

3 -CO2

• Most of CO2 produced during metabolism is hydrated and transported as bicarbonate ion

• Some CO2 is carried as carbamate bound to the uncharged amino group

• Hg-NH2+ CO2 Hb-NH-COO- + H+

• This stabilizes the T –form and release O2 at tissuesbut in the lungs CO2 dissociates from Hb and released in breath.

Effect of 2,3-bisphosphoglycerate on oxygen affinity:

• 2,3- Bisphosphoglycerate (2,3-BPG) is an important regulator of the binding of oxygen to hemoglobin.

• 2,3-BPG is synthesized from an intermediate of the glycolysisintermediate of the glycolysis.

Binding of 2,3-BPG to deoxyhemoglobin (T form):

• 2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin binding to deoxyhemoglobin but not to oxyhemoglobin.but not to oxyhemoglobin.

• This preferential binding stabilizes the T conformation of deoxyhemoglobin.

Response of 2,3-BPG levels to chronic hypoxia or anemia:

The concentration of 2,3-BPG in the red blood cell increases in response to chronic

hypoxia (in certain lung diseases or high altitude) or chronic anemia

(oxygen available to Hb is low in these cases)

• Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of oxygen in the capillaries of tissues.

4 -2,3-bisphosphoglycerate (2,3 BPG)

• Carbon monoxide (CO) binds tightly (but reversibly)tightly (but reversibly) to the hemoglobin iron, forming carboxyhemoglobin (carboxy Hb)carboxyhemoglobin (carboxy Hb)

Dangers of CO binding to hemoglobin (in CO poisoning)Dangers of CO binding to hemoglobin (in CO poisoning)

1- The affinity of hemoglobin for CO is 220 times greater than for oxygen 1- The affinity of hemoglobin for CO is 220 times greater than for oxygen (in availability of both, hemoglobin binds CO more) (in availability of both, hemoglobin binds CO more)

2- When carbon monoxide binds to one or more of the four heme sites, hemoglobin shifts to the relaxed conformation (R-form), causing the remaining heme sites to bind oxygen with high affinity (tightly).

As a result, the affected hemoglobin is unable to release oxygen to the As a result, the affected hemoglobin is unable to release oxygen to the tissues leading to tissues leading to tissue hypoxiatissue hypoxia..

CO poisoning is treated with hyperbaric 100% oxygen therapy to facilitate the dissociation of CO from hemoglobin.

4 -Carbon monoxide (CO)

Factors favoring the T-form of Hb. are:

• Deoxygenation

• Low pH ( H+)

• CO2

• Lactic acid

• 2,3 bisphosphoglycerate

Factors favoring the R-form of Hb. are:

• O2

• CO