Protein FunctionProtein Function

Myoglobin and Myoglobin and HemoglobinHemoglobin

OO22 Binding and Allosteric Properties of Binding and Allosteric Properties of HemoglobinHemoglobin

• Hemoglobin binds and transports HHemoglobin binds and transports H++, O, O22 and COand CO22 in an allosteric manner in an allosteric manner

• Allosteric interactionAllosteric interaction – – of, relating to, undergoing, of, relating to, undergoing, or being a change in the shape and activity of a protein (as an or being a change in the shape and activity of a protein (as an enzyme) that results from combination with another substance enzyme) that results from combination with another substance at a point other than the chemically active siteat a point other than the chemically active site

• a regulatory mechanism where a small a regulatory mechanism where a small molecule(effector) binds and alters an molecule(effector) binds and alters an enzymes activityenzymes activity

Protein FunctionProtein FunctionOO22 does not easily diffuse in muscle and O does not easily diffuse in muscle and O22 is toxic to biological is toxic to biological

systems, so living systems have developed a way around this.systems, 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 Diving animals have large concentration of myoglobin to keep Okeep 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 blood to lungsfrom blood 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))

x-ray crystallography x-ray crystallography of myoglobinof myoglobin

– Made up of 8 Made up of 8 helix A to H helix A to H (proline near (proline near end)end)

– very small due very small due to the foldingto the folding

– hydrophobic hydrophobic residues residues oriented towards oriented towards the interior of the interior of the proteinthe protein

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

– Red indicates Red indicates Heme groupHeme group

Structure of heme prosthetic groupStructure of heme prosthetic group

Protoporphyrin ring w/ iron Protoporphyrin ring w/ iron = heme= heme

Four Pyrrole groups [A to D] Four Pyrrole groups [A to D] linked by methane bridgeslinked by methane bridges

FeFe+2+2 coordinated by coordinated by prophyrin N atoms and a N prophyrin N atoms and a N from Histidine (blue)from Histidine (blue)– This is known as His F8 This is known as His F8

(8(8thth residue of the F residue of the F helix helix

Iron is out of plane due to His 8 Iron is out of plane due to His 8 bondbond

A molecule of OA molecule of O22 acts as 6 acts as 6thth ligandligand

Structure of heme prosthetic groupStructure of heme prosthetic group Two hydrophobic sidechains Two hydrophobic sidechains

on O2 binding site of heme on O2 binding site of heme elp hold it in placeelp hold it in place– Valine E11 and Valine E11 and

phenylalanine CD1phenylalanine CD1

oxygenation changes state of oxygenation changes state of FeFe– Purple to red color of Purple to red color of

blood, Feblood, Fe+3+3 – brown – brown

Oxidation of FeOxidation of Fe+2+2 destroys destroys biological activity of biological activity of myoglobinmyoglobin

Physical barrier of protein is Physical barrier of protein is to maintain oxidation state to maintain oxidation state of Feof Fe+2+2

free vs. bound heme - free vs. bound heme - role of apoproteinrole of apoprotein

ApoproteinApoprotein - - the protein moiety the protein moiety of a molecule or complex of a molecule or complex

restricts heme dimersrestricts heme dimers

keeps iron reduced keeps iron reduced

stabilizes transition state stabilizes transition state (O(O22 binding) binding)

CO, NO and HCO, NO and H22S binding - S binding - poison of Opoison of O22 binding bind binding bind with greater affinity than Owith greater affinity than O22

His E 7 decreases affinity of His E 7 decreases affinity of ligands (CO and Oligands (CO and O22 ) for Fe ) for Fe+2+2

Myoglobin and HemoglobinMyoglobin and Hemoglobin Hemoglobin is structurally related to Hemoglobin is structurally related to

myoglobinmyoglobin very different primary sequence about an very different primary sequence about an

18% homology in the primary sequence18% homology in the primary sequence 2 alpha subunits and 2 beta subunits2 alpha subunits and 2 beta subunits in adults there are very small amount of in adults there are very small amount of

alpha alpha 22delta delta 2 2 hemoglobinhemoglobin

- significance of conserved amino - significance of conserved amino acids between myoglobin and acids between myoglobin and hemoglobinhemoglobin

•these are the important aas these are the important aas which keep hemes in contact with which keep hemes in contact with the proteinthe protein

•Stabilizes helical arraignmentStabilizes helical arraignment•Interacts with heme/ironInteracts with heme/iron

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

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

3D structure of hemoglobin and 3D structure of hemoglobin and myoglobinmyoglobin

11- - 1 1 units have 35 units have 35 interactions interactions

11- - 22 units have 19 units have 19 interaction sitesinteraction sites

similar units have few similar units have few polar contactspolar contacts

the two the two and two and two subunitssubunitsface each face each other through aqueous other through aqueous channelschannels

Binding of oxygen dramatically alters the interactions and brings about a twisting of the two halves (alpha beta pairs)

Much of the quaternary changes takes place in the salt bonds between the C terminals of all four chains

Myoglobin OMyoglobin O22 affinity affinitymyoglobin -oxygen dissociationmyoglobin -oxygen dissociation

K =[Mb] [O2]

[MbO2]

Y =[MbO2] + [O2]

[MbO2]

Y =[O2] + K

[O2]

Y =pO2 + P50

pO2

Y = fraction of globin bound to OY = fraction of globin bound to O22

K = dissociation constantK = dissociation constant

2

MbO2 <-> Mb + O2

Myoglobin vs. Hemoglobin OMyoglobin vs. Hemoglobin O22 affinity affinity

Note the Note the hyperbolic hyperbolic vs sigmoidal vs sigmoidal nature of nature of the two the two curves!curves!

Sigmoidal Sigmoidal curves curves indicate indicate cooperativitcooperativityy

Hemoglobin -sigmoidal dissociationHemoglobin -sigmoidal dissociation

n = Hill constant - determined graphically by the - hill plot n is the slope at midpoint of binding of log (Y/1-Y) vs log

of pO2

if n = 1 then non cooperativityif n < 1 then negative cooperativityif n >1 then positive cooperativity

Hb(O2)n =Hb +n O2

log1 - Y

Y= n log pO2 - n log P50

Xn = number of Hb subunits

Y = fraction of globin Y = fraction of globin bound to Obound to O22

•The experimentally determined The experimentally determined slope does not reflect the number of slope does not reflect the number of binding sites however. It reflects, binding sites however. It reflects, instead, the degree of cooperativity. instead, the degree of cooperativity.

So how does this relate the biological effect of O2 affinity of Hb?

• look at pO2 of alveoli vs. metabolically active tissue• What if oxygen dissociation were hyperbolic rather than sigmoidal?

Alteration in oxygen binding by:Alteration in oxygen binding by:- H+, CO- H+, CO22 2,3 bisphosphoglycerate (BPG) 2,3 bisphosphoglycerate (BPG)

Bohr Effect (CO2 & pH effect on O2 binding)Simple rule of thumb-– High H+ conc and higher COHigh H+ conc and higher CO22 levels decrease O levels decrease O22 affinity for affinity for

HbHb– High OHigh O2 2 concentration decrease H+ and COconcentration decrease H+ and CO22 affinity for Hb affinity for Hb

Hb acts as a H+ buffer in respiring tissue

% O2 Sat

pO2

% O2 Sat

pO2

effect of pH effect of BPG

Cooperative Binding of OCooperative Binding of O22 to Hemoglobin to Hemoglobin

pH = 7.2

pH = 7.4

pH = 7.6

No cooperativity

% S

atur

atio

n

20 40 60 80 100 120pO2 (torr)

BPG and COBPG and CO2 2 Effects on OEffects on O22 Binding Binding

Hb + BPG + CO2

Hb + BPG

Hb + CO2

% S

atur

atio

n

20 40 60 80 100 120pO2 (torr)

Stripped Hb

Functional Structure of Hemoglobin - allosteric

regulations Allosteric interaction - Allosteric interaction - the binding of the binding of

one ligand at one site in a protein that one ligand at one site in a protein that affects the binding of other ligands at affects the binding of other ligands at other sites in the protein. other sites in the protein. This can be This can be affect on binding can be cooperative affect on binding can be cooperative (pos or neg). Allosterism is typically (pos or neg). Allosterism is typically seen when sigmoidal binding / activity seen when sigmoidal binding / activity curves.curves.

Simple definition of the concept of Simple definition of the concept of cooperativity.cooperativity. In its simplest In its simplest meaning, cooperativity is a measure of meaning, cooperativity is a measure of communication between binding sites.communication between binding sites. Positive cooperativity describes a Positive cooperativity describes a relationship in which the binding to one relationship in which the binding to one site facilitates the binding to the second, site facilitates the binding to the second, third, ect.third, ect. No cooperativity puts all sites No cooperativity puts all sites on equal footing and indicates no site to on equal footing and indicates no site to site influence. Negative cooperativity site influence. Negative cooperativity implies binding to one site hinders implies binding to one site hinders binding to subsequent sites.binding to subsequent sites.

So why is this important.So why is this important. Look at the Hill plot and the Look at the Hill plot and the plot of the of binding vs pO2 (figs 7-8 and 7-7 respectively). Think plot of the of binding vs pO2 (figs 7-8 and 7-7 respectively). Think of when hemoglobin should be mostly saturated and when it would of when hemoglobin should be mostly saturated and when it would be best if it had a low saturation / affinity and thus "give up" its be best if it had a low saturation / affinity and thus "give up" its oxygen. Use the table below to help your thoughts.oxygen. Use the table below to help your thoughts.

Oxygen pressure in various fluids

Region or fluid

Inspired Air

Aveolar Air

Arterial Blood

Capilary

Insterstitual Fluid

Cell Cytosol

pO2 (Torr)

158

100

90

40

30

10

Look at the Look at the pOpO22 pressure pressure and think of and think of where Hb where Hb will be will be loaded with loaded with oxygen and oxygen and where it will where it will deliver deliver oxygenoxygen

How is this cooperation actually forced on How is this cooperation actually forced on hemoglobin?hemoglobin?

The structures in one subunit effects the othersThe structures in one subunit effects the others• Structural differences of bound and unbound HbStructural differences of bound and unbound Hb

– Two structures - T and R form depending on the Two structures - T and R form depending on the oxygenation of Hboxygenation of Hb

– T is the deoxy form, R is oxy formT is the deoxy form, R is oxy form– allosteric modifiers shift or stabilize one particular allosteric modifiers shift or stabilize one particular

conformation over the otherconformation over the other– Monomer interactions, most chain interaction occurs Monomer interactions, most chain interaction occurs

between between and and chains chains» through mostly hydrophobic residuesthrough mostly hydrophobic residues and and interactions are few and polarinteractions are few and polar and and contact act as a switch contact act as a switch

oxy and deoxy quaternary structures are different

– change takes place between 1 - 2 and 2 - 1

– amino acids between 1 and 2 help to stabilize each forms

Oxygen binding shifts Oxygen binding shifts quaternary structure at quaternary structure at long distanceslong distances

–binding of O2 ligand at 6th coordinate position pulls Fe into heme

–moves proximal histadine (F8) and the alpha helix it is attached to.

–shift in the helix is transmitted throughout of molecule

– 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.

Note that binding of one or more oxygen can have a dramatic affect on the other subunits that have not yet bound an O2.

How Do Myoglobin (Hemoglobin) Bind How Do Myoglobin (Hemoglobin) Bind to Oxygento Oxygen

NNH

His E7

Move downupon O2 binding

How Do Myoglobin (Hemoglobin) Bind How Do Myoglobin (Hemoglobin) Bind to Oxygento Oxygen

NNH

His E7

Move downupon O2 binding

T and R State of Hemoglobin - ReviewT and R State of Hemoglobin - Review Below are the two major conformations of hemoglobin as predicted by the models Below are the two major conformations of hemoglobin as predicted by the models

for allosteric activation. for allosteric activation. Oxygen will bind to hemoglobin in either state; however, it has a signficantly Oxygen will bind to hemoglobin in either state; however, it has a signficantly

higher affinity for hemoglobin in the R state.higher affinity for hemoglobin in the R state. In the absence of oxygen, hemoglobin is more stable in the T state, and is In the absence of oxygen, hemoglobin is more stable in the T state, and is

therefore the predominant form of deoxyhemoglobin. R stands for relaxed, while therefore the predominant form of deoxyhemoglobin. R stands for relaxed, while T stands for tense, since this is stabilized by a greater number of ion pairs. T stands for tense, since this is stabilized by a greater number of ion pairs.

Upon a conformational change from the T state to the R state, ion pairs are Upon a conformational change from the T state to the R state, ion pairs are broken mainly between the broken mainly between the 1122 subunits. subunits.

Structural Changes when Structural Changes when converting R to T Hbconverting R to T Hb

Bottom Line:Bottom Line:

COCO22, BPG and H, BPG and H++ all help to all help to stabilize Hb in R or T form.stabilize Hb in R or T form.

Bohr Effect ContinuedBohr Effect ContinuedThe Bohr effect is the reversible shift in Hb affinity for OThe Bohr effect is the reversible shift in Hb affinity for O22 with changes in with changes in pH.pH.

H+ Transport (effect) -H+ Transport (effect) - O O22 binding to Hb releases H binding to Hb releases H++ due to conformational due to conformational changes in Hbchanges in Hb- - deoxyform (T form) brings Asp 94 close to His 146 (fig 7-11 (b))deoxyform (T form) brings Asp 94 close to His 146 (fig 7-11 (b))- the proximity of an acidic amino acid increases the pK of histidine (pKa - the proximity of an acidic amino acid increases the pK of histidine (pKa is now above the pH) and results in His now above the pH) and results in H++ “binding” to deoxyHb - in other “binding” to deoxyHb - in other words the His becomes protonated where it normally would be ionizedwords the His becomes protonated where it normally would be ionized

- increasing pH stimulates Hb to bind to Oincreasing pH stimulates Hb to bind to O22

- Bottom line - when OBottom line - when O22 binds Hb, H+ is released from several amino binds Hb, H+ is released from several amino acid's functional groups. When Oacid's functional groups. When O22 is released, the amino acids become is released, the amino acids become protonized and then "picks" up a H+.protonized and then "picks" up a H+.

So when the H+ is high (acidic conditions) the H+ is driven onto the So when the H+ is high (acidic conditions) the H+ is driven onto the terminal amino acids driving it into the T conformationterminal amino acids driving it into the T conformation

COCO22 effects effects - In the red blood cells the picture is even - In the red blood cells the picture is even

more complicated. COmore complicated. CO22 is removed by converting CO is removed by converting CO22 to to bicarbonatebicarbonate

CO2 + H2O <-> HCO3- + H+

bicarbonate (HCObicarbonate (HCO33--) formed by the enzyme carbonic ) formed by the enzyme carbonic

anhydraseanhydrase–H+ produced by this enzyme is removed by the Hb as H+ produced by this enzyme is removed by the Hb as described abovedescribed above

–This allows more COThis allows more CO2 2 to be removed in the form of to be removed in the form of bicarbonatebicarbonate

COCO22 binding aids in reducing O binding aids in reducing O22 affinity by affinity by changing conformation by the production changing conformation by the production of more H+ (R to T change)of more H+ (R to T change)

In the lungs the OIn the lungs the O22 binds Hb and binds Hb and forces the R conformationforces the R conformation

This all aids the function of Hb. In active tissues respiration, (glycolysis) results in lactic acid formation. These tissues need more O2. Without the H+ effect Hb would hold on to more of the O2. The H+ induces Hb to dump 10% more of it's O2.

– CO2 reversibly binds to N term (carbamate) to remove remaining CO2

R - NH2 + CO2 <-> R - NH - COO- + H+

R is the Hb N term amide

The carbamide increases the T formation - deoxy form. The reverse occurs in the lungs. This results in 1/2 of CO2

removal from tissues.

O

C

O

C C

R

H

O

ProteinH2N

Amino Terminus

C C

R

H

O

ProteinHN

Carbamate on Amino Terminus

C

O

O

H+

2,3 2,3 bisphosphoglycerate bisphosphoglycerate (BPG) Effects(BPG) Effects

– Purified Hb has a different O2 affinity than it does in blood

26 fold decrease change in affinity is due to 2,-3 diphosphoglycerate BPG

diphosphoglycerate BPG

present in human red blood cells at present in human red blood cells at approximately 5 mmol/L. approximately 5 mmol/L.

It binds with greater affinity to deoxygenated It binds with greater affinity to deoxygenated hemoglobinhemoglobin

In bonding to partially deoxygenated In bonding to partially deoxygenated hemoglobin it allosterically upregulates the hemoglobin it allosterically upregulates the release of the remaining oxygen molecules release of the remaining oxygen molecules bound to the hemoglobin, thus enhancing the bound to the hemoglobin, thus enhancing the ability of RBCs to release oxygen near tissues ability of RBCs to release oxygen near tissues that need it most that need it most

2,3 bisphosphoglycerate (BPG)– Purified Hb has a different O2

affinity than it does in blood 26 fold decrease change in

affinity is due to 2,-3 diphosphoglycerate BPG– (BPG replaced by

nucleotides IHP and ATP in fish and birds)

– - 1 BPG per Hb - binds in central cavity of Hb

– - binds preferentially to deoxy Hb

– - hydrophobic bonds with Lys and salt bridge with His

– - O2 binding changes conformation and “kicks out” BPG

– change in altitude increases concentration of BPG

Fetal F Hb has replaced His 143 with Ser - What might the consequences be?

Cooperative interactions between Cooperative interactions between subunitssubunits

Both models do not fully account for the effects of allosteric effectors– sequential model (D Koshland)

binding of one O2 induces T-R conformation change 1st change is most difficult due to influence by 3 other

subunits binding of next three subunits happens sequentially, with

higher affinity (easier T-R changes) kinetics increase to the fully oxy Hb state as more O2 is

bound

Concerted model (J Monod) All R or all T no in between as in the All R or all T no in between as in the

Koshland modelKoshland model concerted model means as more O2

binds, the R conformation is favored until all units are in the R conformation regardless of the total units bound to O2

Affinities do NOT change until conformation changes

1 O2 - all T; 2 O2 - nearly even equilibrium; 3 O2 mostly R; 4 O2 - mostly R form

energy from O2 binding causes the change in equilibrium

this model best fits O2 dissociation curve but with limits.

Sickle-Cell Anemia, a Molecular Sickle-Cell Anemia, a Molecular DiseaseDisease

One of the first “molecular” diseases found - sickle cell anemia– sickle cell - blood cell is elongated , mis-shaped

(sickle) occurs at low O2 concentration caused by hemoglobin aggregates inflammation in capillaries and pain red blood cells break down - anemia

– between 10% of American blacks and 25% of African blacks are heterozygous for sickle cell anemia

– homozygous usually do not survive into adult hood– heterozygous individuals usually have no problem

except when in severe oxygen deprivation

Single amino acid (point mutation) HbS vs. HbA changes structure– sickle cell b chains have a valine in place of glutamate– leads to more Hb S (sickle cell) has 2 more + charges

than normal hemoglobin Glu -Val occurs on exterior of protein - does not

change O2 dissociation/allosteric properties of protein

Deoxy HbS precipitatesDeoxy HbS precipitates– oxyHb phenylalanine b85 oxyHb phenylalanine b85

and leucine b88 interiorand leucine b88 interior– phe and leu shift to exteriorphe and leu shift to exterior

– create a sticky patch with create a sticky patch with valine (hydrophobic valine (hydrophobic bonding)bonding)

– nucleation (cluster of nucleation (cluster of aggregate) occurs aggregate) occurs logarithmicallylogarithmically– homozygous - 1000 times homozygous - 1000 times

faster than heterozygousfaster than heterozygous– that means mixed genes that means mixed genes

can re-oxygenate faster can re-oxygenate faster than polymerization can than polymerization can occuroccur

how can such a disease occur?– highest concentration of gene mutation

occurs where there is high incidence of malaria

– heterozygous individuals survive this disease better than those without

– malaria causing parasite lives in red blood cells during part of its life cycle

– partial sickling must interrupt life cycle of malaria parasite

Any Questions?Any Questions?