Emoglobina e Mioglobina

The structure-function relationship

Protein sequence (1 structure) determines 3-D structure (3, 4), and 3-D structure determines function. n addition to structure, d!namics (motions) of "iomolecules also pla! a role in determinin# function.

$unction% o&!#en carrier

The pro"lem% '&!#en is required "! cells throu#hout or#anisms for respiration. (ut)

* Diffusion of o&!#en throu#h tissues is ineffecti+e. * '&!#en has lo, solu"ilit! in aqueous solutions.

To sol+e this pro"lem, ,e need a carrier molecule that can transport o&!#en from the lun#s to tissues ,here the o&!#en is needed.

The solution% hemo#lo"in

-emo#lo"in has four heme (ironporph!rin) prosthetic #roups that "ind o&!#en. The protein surroundin# the hemes pla!s important roles. Pre+ents side-reactions li.e irre+ersi"le heme o&idation /nhances selecti+it! for '0 "indin# (relati+e to 1' for e&ample) Tetramer allo,s for cooperati+it! ,hich ma.es hemo#lo"in efficient.

-emo#lo"in function

First Protein Structure Myoglobin. Protein purified from whale blood. Max Perutz 1958. Showed a 75% -helical fold. - 155 amino acids, ~ 17 kDa.

First Protein Complex Hemoglobin. Two copies each of & chains of myoglobin in a complex. Solved by John Kendrew.

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

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

Properties of a "iolo#ical o&!#en carrier

(inds o&!#en re+ersi"l!

* /fficientl! pic. up o&!#en in the lun#s (hi#h capacit! and fast "indin#) * /fficientl! release o&!#en in tissues ,here it is needed * 2o side reactions 3etals are "etter candidates for re+ersi"le o&!#en "indin# than or#anic molecules (ut ) reaction of 4free4 metals ,ith o&!#en can "e irre+ersi"le and can also lead to production of dan#erous free radicals.

5i#ands in "iolo#!

'&!#en is a li#and for hemo#lo"in. 6 li#and is a small molecule that "inds specificall! to a lar#er one. /&amples% -ormones, su"strates, anti#ens. 7ater can "e a li#and, as lon# as it "inds specificall!. (3ost ,ater-"iomolecule interactions are nonspecific).

5i#and affinit!

5i#and "indin# is a reaction, and li#and affinit! can "e descri"ed usin# an equili"rium constant%

P+L

PL

Keq = [PL] [P] [L] = Ka (association constant, 3 Keq = [P] [L] [PL] = Kd (dissociation constant, 3)
-1)

(not to "e confused ,ith acid dissociation constant)

PL

P+L

8d is in more common use.

5i#and affinit!

8d is 95: for half dissociation%.

5i#and affinit!

8d is a measure of "indin# stren#th%.

5i#and affinit!

8d for a #aseous li#and ma! "e reported as a partial pressure. $or the '0 stora#e protein m!o#lo"in (3"), for e&ample%

Mb-O2

Mb + O2

Kd = [Mb] pO2 [MbO2]

?nits are .Pa, or mm-#
;<01<=>

$i#. >-4"

Fraction binding sites occupied

-eme is the li#and "indin# site

The heme #roup. Porph!rin occupies 4 of the @ coordination sites of the iron%

The #lo"in fold

2ote%
/i#ht -helices (6--) connected "! turns and loops. -eme "inds "et,een helices / and $. -istidine residues from helices / and $ are situated near the heme iron.

$i#. >-3

;<01<=>

'&!#en-"indin# site
'ne -is residue coordinates the iron. The second one assists ,ith sta"iliAin# the '0-"ound form and also desta"iliAin# the 1'"ound form. 1' "indin# onl! 0==& stron#er (compare to 0=,===& to free heme).

3!o#lo"in
p'0 in lun#s

p'0 in tissues

3!o#lo"in is an o&!#en stora#e protein in muscles. 1onsider - ,h! is it not a #ood o&!#en transport proteinB

-emo#lo"in structure

3!o#lo"in is a monomer. /ach hemo#lo"in protomer resem"les m!o#lo"in%

-emo#lo"in structure

Ctron#est su"unit interactions are hi#hli#hted%

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!

-emo#lo"in d!namics

'&!#en "indin# tri##ers local conformational chan#es%

$i#. >-11

The shift in the iron tu#s on the -is, ,hich mo+es heli& $

-emo#lo"in d!namics

-emo#lo"in d!namics

6nd the motion of heli& $ is translated to #lo"al conformational chan#es%

1HGA

1BBB

onic interactions "et,een su"units

These interactions sta"iliAe the T state. The! are disrupted upon o&!#en "indin#, promotin# transition to the D state.

Structure of Hemoglobin

Water bound to heme Iron

Oxygen bound to heme Iron

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 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

onic interactions "et,een su"units

These interactions sta"iliAe the T state. The! are disrupted upon o&!#en "indin#, promotin# transition to the D state.

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

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

0,3-"isphospho#l!cerate "indin#

(indin# of 0,3-(PE - "inds to poc.et in center of tetramerF "ut onl! in the T state.

0,3-(PE increases hemo#lo"in efficienc! "! sta"iliAin# the T state

Oxygen affinity of fetal & maternal red blood cells

Fetal Hgl does not bind 2,3-BPG, higher O2 affinity Fetal hemoglobin tetramer has 2 alpha & 2 gamma chains, Gene duplication

27

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

1ooperati+it! leads to alloster!

Deo&!hemo#lo"in lo,-affinit! Tstate pic.s up '0 in the lun#s, ,here p'0 is hi#h, 1on+ertin# it into the hi#haffinit! D-state, so it readil! pic.s up more '0 6s loaded o&!hemo#lo"in (Dstate) mo+es "ac. to tissues, ,here p'0 is lo,er, it "e#ins to lose its '0, 7hich facilitates the transition "ac. to the T-state 7hich facilitates loss of '0)

6lloster! is necessar! for function

1ompare to 3"%

-emo#lo"in also carries 1'0 and -G

The amino-termini of the four chains "ecome car"am!lated at lo, p- (H.0) in the tissues -elpin# to transport a"out 0=I of the car"on dio&ide formed in the tissues to the lun#s and .idne!s (ut ,hat happens to the rest of the 1'0B )and ho, else is the Jp-K in+ol+edB

Decall aqueous 1'0 forms car"onic acid

1ar"on dio&ide released in tissues is insolu"le until h!drated and con+erted to "icar"onate "! the enA!me car"onic anh!drase (ut this releases -G (causin# the p- in the tissues and "lood to fall) This process is re+ersed in the lun#s )

Dole of -" in carr!in# -G

-istidine -13 of T-state hemo#lo"in has a hi#her p8a than normal (+ia interaction ,ith 6sp $E1), and thus #ets readil! protonated (ac. at the lun#s, return to the D-state shifts -13 "ac. to its normal p8a, releasin# the -G Delease of -G promotes con+ersion to the D state, increasin# '0 affinit!)

The (ohr /ffect

/ffect of p- and p1'0 on -" o&!#en affinit!

deo&!-" (T-state)

The (ohr /ffect

This Jeffect of p- and 1'0 concentration on the "indin# and release of o&!#en "! hemo#lo"inK is thus dependent on The allosteric chan#es in the tetramer that interrelate the ph!siolo#ical parameters in tissues, "lood, and lun#s 7ith the a"ilit! to "ind o&!#en, 6nd carr! "oth car"on dio&ide and protons a,a! from tissue and "ac. to the lun#s.

0,3-"isphospho#l!cerate "indin#

(indin# of 0,3-(PE - "inds to poc.et in center of tetramerF "ut onl! in the T state.

0,3-"isphospho#l!cerate "indin#

0,3-(PE concentration in "lood is > m3 at sea le+el, "ut L m3 at hi#h altitudes )

3odel for cooperati+e "indin#

Cequential model%

(indin# of a li#and to a su"unit affects the affinit! of the other su"units for the li#and. n terms of "indin# constants% 81 M 80 M 83 M 84. There also is a 4concerted model.4 (oth to#ether pro+ide a description of the process.

6lloster!% "indin# of 5 to one su"unit affects affinit! of other su"units.

Models for Allosteric Behavior

Monod, Wyman, Changeux (MWC) Model: allosteric proteins can exist in two states: R (relaxed) and T (tense) In this model, all the subunits of an oligomer must be in the same state T state predominates in the absence of substrate S S binds much tighter to R than to T

More about MWC

Cooperativity is achieved because S binding increases the population of R, which increases the sites available to S Ligands such as S are positive homotropic effectors Molecules that influence the binding of something other than themselves are heterotropic effectors

Cic.le-cell anemia

Cic.le-cell anemia

6 #enetic disease resultin# from a mutation that con+erts Elu@ (acidic) in the -chains to Nal (nonpolar). This su"stitution creates h!dropho"ic Jstic.!K patches on the normall! char#ed surface of the chains.

The o&!#enated molecules are solu"le, "ut upon deo&!#enation, the conformation of -"C differs considera"l! from -"6, and it a##re#ates into insolu"le fi"ers (as dia#rammed on the ne&t slide). These fi"ers deform the D(1s into spin! or sic.leshaped cells.

Cic.le-cell anemia

Fig. 7-18b

8e! points a"out -" and li#and "indin#

$or a monomeric protein li.e m!o#lo"in, the simple re+ersi"le "indin# of o&!#en ma! "e descri"ed "! a dissociation constant, 8D Proteins ma! chan#e conformation ,hen li#ands "ind (alloster!)F in a multimer, other su"units can "e affected -emo#lo"in is an 00 heterotetramer, in ,hich o&!#en "indin# increases the sta"ilit! of the D-state relati+e to the T-state -emo#lo"in also "inds -G and 1'0, formin# ion pairs that lessen the affinit! for o&!#en ((ohr effect)F the "indin# of 0,3-"isphospho#l!cerate also pre+ents efficient o&!#en "indin# 6 #enetic mutation of Elu@ to Nal causes sic.le-cell anemia "ecause the h!dropho"ic patch that leads to a##re#ation of the deo&!hemo#lo"in form.