By: Khuram Aziz M Phill Biochemistry Junior scientist By IBC Life sciences Member of NAYS

2

• Determined by a variety of bonding interactions between the "side chains" on the amino acids:

– Hydrogen bonds

– Disulfide bonds

– Non-polar interaction

– Salt bridgesE Obayashi et al. Nature 2008

• non-linear• 3 dimensional• global but restricted to the

amino acid polymer• formed and stabilized by

hydrogen bonding, covalent (e.g. disulfide) bonding, hydrophobic packing toward core and hydrophilic exposure to solvent

• A globular amino acid polymer folded and compacted is somewhat functional (catalytic) and energetically favorable interaction!

Small protein found in Small protein found in musclemuscle

Made up of 153 residues Made up of 153 residues grouped into 8 grouped into 8 helix A to helix A to H (proline near end)H (proline near end)

very small due to the very small due to the foldingfolding 44 x 44 x 25 Å44 x 44 x 25 Å

hydrophobic residues hydrophobic residues oriented towards the oriented towards the interior of the proteininterior of the protein

only polar AAs inside are 2 only polar AAs inside are 2 histidineshistidines

Hemoglobin and Hemoglobin and myoglobin are only myoglobin are only slightly related in primary slightly related in primary sequence. sequence.

Although most amino Although most amino acids are different acids are different between the two between the two sequences, the amino sequences, the amino acid changes between acid changes between the two proteins are the two proteins are generally conservative. generally conservative.

More strikingly, the More strikingly, the secondary structures of secondary structures of myoglobin and the myoglobin and the subunits of hemoglobin subunits of hemoglobin are virtually identical.are virtually identical.

• non-linear• 3 dimensional• global, and across

distinct amino acid polymers

• formed by hydrogen bonding, covalent bonding, hydrophobic packing and hydrophilic exposure

• favorable, functional structures occur frequently and have been categorized

• Globular: – Folded in a globular shape– Same or different types of

secondary structure– Example: haemoglobin

• Fibrous: – Polypeptides arranged in long

strands or sheets– One type of secondary

structure– Examples: collagen and silk

Hemoglobin

Collagen

Quaternary structure involves several polypeptides: Oligomers Heteromers

These subunits interact with each other through the usual weak interaction forces (H bonds, Van der Waals, ionic interactions, hydrophobic interactions) and/or though disulfide bonds;

For aquous proteins, frequently, but not always, the interface between two subunits is made of hydrophobic amino acids.

For membrane-bound proteins, the amino acids at the interface between the subunits are usually hydrophilic;

9Porin: a trimeric membrane-bound protein

hydrophobic

hydrophilic

Haemoglobin StructureHaemoglobin Structure

•four-chained protein •oxygen carrying ability •dependant on Hb structure

Made up of 4 polypeptide chains: 2 copies of -subunit (or

HbA): yellow and blue; 2 copies of -subunit (or

HbB): red and pink

Each subunit binds its own heme group: so each subunit can bind O2

Each subunit is highly similar in structure to myoglobin;

Both hemoglobin and myoglobin bind O2 in a very similar fashion

.

OO22 does not easily diffuse in muscle and O does not easily diffuse in muscle and O22 is toxic to biological systems, is toxic to biological systems, so living systems have developed a way around this.so living systems have developed a way around this.

Physiological roles of:Physiological roles of: MyoglobinMyoglobin

Transports OTransports O22 in rapidly respiring muscle in rapidly respiring muscle Monomer - single unitMonomer - single unit Store of OStore of O22 in muscle high affinity for O in muscle high affinity for O22

Diving animals have large concentration of myoglobin to keep ODiving animals have large concentration of myoglobin to keep O2 2

supplied to musclessupplied to muscles HemoglobinHemoglobin

Found in red blood cells Found in red blood cells Carries OCarries O22 from lungs to tissues and removes CO from lungs to tissues and removes CO22 and H and H++ from from

blood to lungsblood to lungs Lower affinity for OLower affinity for O2 2 than myoglobinthan myoglobin

Tetrameter - two sets of similar units (Tetrameter - two sets of similar units (2222))

Myoglobin and Hemoglobin are oxygen carrying molecules that overcome the problem that vertebrates have with the low solubility of oxygen in water

O2 O2 O2

Hemoglobin serves as the carrier ofoxygen in blood AND also aids in thetransport of carbon dioxide and H+

Myoglobin provides muscle tissue withan oxygen reserve AND facilitates oxygenmovement in muscle

Haemoglobin

Myoglobin

1. Found in the blood.

2. It joins to 4 molecules of oxygen at its maximum saturation but low affinity compared to myoglobin.

3. The haemoglobin never

reaches 100% oxygen saturation

1. Found in Muscles and tissues

2. It binds to only one molecule of oxygen but there is greater affinity to oxygen.

3. The myoglobin can reach 100% oxygen saturation.

15HbA Myoglobin

O2 binds the Fe2+ atom of the heme group, and is held in place with His 64;

Oxygen-bound myoglobin/Hb is called oxymyoglobin/oxyHb

Oxygen-free myoglobin/Hb is called deoxymyoglobin/deoxyHb

Now,

4 major residues surround the heme group: Phe 43 His 64 Val 68 His 93

These amino acids create a hydrophobic environment while help hold the heme group in place;

Also: His 93 binds the Fe2+ atom;

Oxygen binding to hemoglobin is due to the effect of the ligand-binding state of one heme group on the ligand-binding affinity of another.

Too far apart to interact! (25 to 37 Å apart)

Mechanically transmitted between heme groups by motions of the proteins

This means the molecule changes shape!

PDB ID 1HGA

In the deoxyHb form, Fe2+ is bonded to 5 ligands: His 93 and 4 amines from the heme group;

When one subunit of Hb binds O2, the Fe2+ atom moves foward the plane of the heme group, pulling with it the His 93 and the -helix;

This causes a slight but significant change in the tertiary structure of all the other Hb subunits, even if they are in the deoxyHb form;

The consequence of this slight change in conformation is an increase in the affinity of these other Hb subunits for O2;

This phenomenon, where a change in the shape in one subunit trigger similar changes in other subunits of the same molecule, is called cooperativity;

Molecules exhibiting cooperativity are also called allosteric molecules;

CHMI 2227 - E.R. Gauthier, Ph.D. 21http://upload.wikimedia.org/wikipedia/commons/0/07/Hb-animation2.gif

T (Low Affinity) R (High Affinity)

• There are two general structural states There are two general structural states - the deoxy or - the deoxy or T form and the oxy or R form.T form and the oxy or R form.

One type of interactions shift is the polar bonds One type of interactions shift is the polar bonds between the alpha 1 and the beta 2 subunits.between the alpha 1 and the beta 2 subunits.

The two states

Below are the two major conformations of hemoglobin as Below are the two major conformations of hemoglobin as predicted by the models for allosteric activation. predicted by the models for allosteric activation.

Oxygen will bind to hemoglobin in either state; however, it Oxygen will bind to hemoglobin in either state; however, it has a signficantly higher affinity for hemoglobin in the R has a signficantly higher affinity for hemoglobin in the R state.state.

R stands for relaxed, while T stands for tense,R stands for relaxed, while T stands for tense, In the absence of oxygen, hemoglobin is more stable in the T state, and is therefore the In the absence of oxygen, hemoglobin is more stable in the T state, and is therefore the

predominant form of deoxyhemoglobinpredominant form of deoxyhemoglobin Upon a conformational change from the T state to the R state, ion pairs are broken Upon a conformational change from the T state to the R state, ion pairs are broken

mainly between the amainly between the a11bb22 subunits. subunits. Note that binding of one or more oxygen can have a dramatic affect on the other subunits

that have not yet bound an O2.

The T form finds the terminals in several important H bonds and salt bridges.

In the T form the C terminus of each subunit are "locked" into position through several hydrogen and ionic bonds.

Shifts into the R state break these and allow an increased movement throughout the molecule.

Reveals the amount of haemoglobin saturation at different PO2 values.

0 2 4 6 8 10 120

20

40

60

80

100

Oxygen Dissociation Curve for MYOGLOBIN

Myoglobin is a molecule in muscles that combines with oxygen. The oxygen dissociation curve for myoglobin is far to the left of haemoglobin.

Myoglobin is a molecule in muscles that combines with oxygen. The oxygen dissociation curve for myoglobin is far to the left of haemoglobin.

MyoglobinMyoglobin

HaemoglobinHaemoglobin

Sat

urat

ion

of H

aem

oglo

bin

/ %

At high O2 concentrations, both myoglobin and Hb are saturated, meaning there are no more O2-binding spots available.

Interestingly: the affinity of myoglobin and Hb for oxygen varies by a factor of 10: Only 2.8 Torr are required

to get 50% of myoglobin saturated;

However, 26 Torr are required to half-saturate Hb.

A

B

PO2 (mmHg) % saturation of haemoglobin

10 13.5

20 35.0

30 57.0

40 75.0

50 83.5

60 89.0

70 92.7

80 94.5

90 96.5

100 97.5

Table 1. the percentage saturation of haemoglobin with oxygen at different partial pressures of oxygen

Plot a graph of PO2 against the percentage saturation of

haemoglobin. The curve obtained is called the oxygen haemoglobin dissociation curve.

The Bohr effect concerns the observed decrease in O2 binding by hemoglobin when the pH is lowered;

This effect explains why hemoglobin binds O2 in the lungs, and releases it in the tissues;

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w.aw

-bc.com/m

athews/ch07/fi7p16.htm

Blood in lungs has higher pH than blood in capillaries of metabolic tissues

Affinity for oxygen depends on the pH

Oxygen binds well at higher pH

Oxygen is released well at lower pH

The pH difference between lungs and metabolic tissues increases the O2 transfer

efficiency

This is known as the Bohr effect

33TissuesErythrocytes

Glucose + O2

ATP

CO2

CO2H2O

Carbonic anhydrase

H2CO3

HCO3-

H+

Plasma

HCO3-

(to lungs)

Hb-4O2

Hb-H+

4O2 4O2

H2O + Cl-

34LungsErythrocytes

CO2H2O

Carbonic anhydrase

H2CO3

HCO3-

H+

Plasma

HCO3-

Hb-4O2

Hb-H+

4O2 O2

H2O + Cl-

CO2

Air

CO2

O2

Lungs at sea level: PO2 of 100mmHg haemoglobin is 98% SATURATED

Lungs at high elevations: PO2 of 80mmHg, haemoglobin 95% saturated

Even though PO2 differs by 20 mmHg there is almost no difference in haemoglobin saturation.

When the PO2 in the lungs declines below typical sea level values, haemoglobin still has a high affinity for O2 and remains almost fully saturated.

PH

• lowering of blood pH (making blood more acidic)

• caused by presence of H+ ions from lactic acid or carbonic acid

• reduces affinity of Hb for O2

• and more O2 is delivered to acidic sites which are working harder

TEMPERATURE increased blood temperature reduces haemoglobin affinity for O2

hence more O2 is delivered to warmed-up tissue

CARBON DIOXIDE CONCENTRATION• the higher CO2 concentration in tissue

• the less the affinity of Hb for O2

• so the harder the tissue is working, the more O2 is released

CO has similar size and shape to O2; it can fit to the same binding site

CO binds over 20,000 times better than O2 because the carbon in CO has a filled lone electron pair that can be donated to vacant d-orbitals on the Fe2+

Protein pocket decreases affinity for CO, but is still binds about 250 times better than oxygen

CO is highly toxic as it competes with oxygen. It blocks the function of myoglobin, hemoglobin, and mitochondrial cytochromes that are involved in oxidative phosphorylation

Autopsy photo showing characteristic skin discoloration

Myoglobin’s affinity for carbon monoxide is ~ 60x its affinity for O2.

Hemoglobin’s affinity for carbon monoxide is ~ 230x its affinity for O2.

Myoglobin is a molecule in muscles that combines with oxygen. The oxygen dissociation curve for myoglobin is far to the left of haemoglobin.

What does this mean? if both Myo and Hb can bind O2, why is it

that Hb is a multimeric protein, while myoglobin is monomeric??? WHY????

Why Hb is allosteric, while Myoglobin is not?