Heme Elek

Dioxygen: Uptake, Transport & Storage:
Hemocyanin/Hemerythrin
Hemoglobin/Myoglobin
References:
M. F. Perutz et al. Acc. Chem. Res. (1987) 20, 309—321.
J. M. Riefkind Adv. Inorg. Biochem. (1988) 7, 155—241.
M. F. Perutz Annu. Rev. Physiol. (1990) 52, 1—25.

M. F. Perutz Ann. Rev. Biophysics and Biomolecular Structure 1998
R. E. Dickerson, I. Geis Hemoglobin 1983
D. Voet & J. G. Voet Biochemistry 2nd ed. 1995
L. Stryer Biochemistry 4th ed. 1997 050902
Dioxygen: Uptake, Transport & Storage Why do we have to bind O2 in first place?
Why not molecular diffusion?
Despite the huge body of synthetic work, NO model system At 100-mm O2 pressure found in the lungs, 0.3 mL of O2 can dissolve per
comes even close to the biological parent! 100 mL plasma…far too little to keep you going.
One would have to breath pure O2 at 3 atm pressure for the solubility to rise
It’s not only about reversible dioxygen binding! to an acceptable 7 mL O2/100 mL plasma…and that’s just enough to enable
you to read this. In contrast, 15g of Hemoglobin (found in 100 mL of
1. The transport molecule must have a high affinity for O2 in the
blood plasma) are capable of binding 20 mL of gaseous O2..and that’s also
presence of plentiful supply at the lungs and a lowered affinity in why giant man-eating killer ants will only exist in Hollywood!
the O2—poorer environment of the muscles…where it is needed!
2. The storage molecule must have a higher affinity for dioxygen What it needs to do “the job”:
than the carrier has at low dioxygen concentrations.
Every mL of blood has approx. 5 billion red blood cells (erythrocytes)
3. The carrier should not only bind to O2, but to carbon dioxide as
…each erythrocyte is packed with 280 million molecules of hemoglobin.
well to transport CO2 back to the lungs where it can be ejected as
a waste product.
4. The carrier should release its dioxygen more readily to working Dickerson & Geis: “If an erythrocyte were enlarged 300 million times,
muscles than to resting tissue. it would be the size, and roughly the shape, of the Rose Bowl, piled with
280 million large grapefruits”.
Dioxygen: Uptake, Transport & Storage Dioxygen: Uptake, Transport & Storage
The Dioxygen-Carrying Team: Hemoglobin/Myoglobin
1
Myoglobin/Hemoglobin Comparison: Size:
Myoglobin:
Only 1 heme and 1 polypeptide chain
of 153 amino acids. MW = 17,800
Hemoglobin:
4 heme units and 4 polypeptide chains;
2 α chains with 141amino acids, and
2 β chains with 146 amino acids.
MW = 64,500
7.5 microns
taken from: Dennis Kunkel Microscopy, Inc.
Myoglobin/Hemoglobin: The first three-dimensional structures Myoglobin: The first three-dimensional structure analysis
of any protein: Nobel Prize 1962 of any protein. Kendrew at al. Nature (1960), 185, 422.
John C.Kendrew
(born 1917, died 1998)
Max Peruz
(born 1914, died 2002)
The richest source of

myoglobin are the muscles of
aquatic diving mammals:
seals, whales, and porpoises.
It is no surprise that the first x-

ray crystal structure of
myoglobin was obtained from
the protein isolated from sperm
whale.
A Three-Dimensional Fourier Synthesis at 2 Å Resolution. A Three-Dimensional Fourier Synthesis at 2 Å Resolution.
Observed (left) and computed (middle) electron density
distribution in the plane of the heme. Photograph of a set of
sections normal to the plane of the heme group (right).
taken from: Kendrew at al. Nature (1960), 185, 422.
…requires an awful lot of imagination!
2
…a note on the side: Science & Art
Geis (1908-1997) was one of the greatest scientific
artists of the 20th century. His innovations, particularly
in depicting the structures of biological macromolecules
such as DNA, earned him an international reputation.
Many of his illustrations appeared in Scientific American,
including a painting of the first protein crystal structure,
of myoglobin, published in 1961.
©2000 Howard Hughes Medical Institute
Myoglobin: Shapes & Perspectives:
an oblate spheroid: 44 x 44 x 25 Å
Front view: Side view:
“Little can be said as yet about the relation between structure and function.
• a box for the heme group, • helices range in length from 7 The haem groups are much too far apart for the combination with oxygen of
• built up from 8 connected, (C,D) to 26 (H) residues. any one of them to affect the oxygen affinity of its neighbours directly.
• right-handed pieces of α helix. • hydrophilic groups outside. Whatever interaction between the haem groups exists must be of subtle and
• hydrophobic groups inside. indirect kind that we cannot yet guess.” M. F. Perutz, Nature 1960, 185, 416.
Size: Hemoglobin: Structures of Deoxy- (left) and Oxyhemoglobin (right)
M. F. Perutz at al. Nature (1960), 185, 416.
2 α and 2 β polypeptide chains. Each of the four chains is folded in much the same
way as seen for Myoglobin. The α1β1 and α2β 2 interactions (35 residues) are
hydrophobic and stronger than α1α2 and β1β2. They are also stronger than the α1β 2
(19 residues).
3
Hemoglobin: Symmetry & Perspective: The quaternary structural changes preserve Hb’s two-fold symmetry
and takes place entirely across the α1β2 and α2β1 interface:
Side view:
Nearly 2,2,2 symmetry (pseudo D2) Oxygenation rotates the α1β1 dimer ~ 15o with respect to the α2β 2 dimer (page 33),
so that some atoms at the α1β2 interface shift by as much as 6 Å relative to each
roughly spherical: 64 x 55 x 50 Å
other.
Myoglobin/Hemoglobin: A comparison Myoglobin/Hemoglobin: A comparison of the amino acid
sequences.
The similarity of their conformations is evident…
Myoglobin Hemoglobin (β-chain)
…yet unexpected, because their amino acid sequences are rather different!
² Different amino acid sequences can specify very similar 3-D structures. the three chains are identical at
only 24 of 141 position.
The interior consists almost entirely of non-polar residues such as
Highly conserved amino acid residues in hemoglobins: leucine (Leu), valine (Val), phenylalanine (Phe), and methionene.
Position Amino Acid Role

F8 Histidine Proximal heme-linked His
E7 Histidine Distal His near the heme
CD1 Phenylalanine Heme contact
F4 Leucine Heme contact
B6 Glycine Allows close approach of the B and
E helices
C2 Proline Helix termination
HC2 Tyrosine Cross-links the H and F helices
C4 Threonine ?
H10 Lysine ?
Additionally, highly non-polar interior and polar character of the exterior The only polar residues are
of the molecule is conserved. two histidines with their critical
function at the binding site.
4
The Role of the Proximal (F8) & Distal (E7) Histidine:
The Role of the Proximal (F8) & Distal (E7) Histidine:
The imidazole ring of the distal His

E7 (pK = 5.5 ) acts a a proton trap,
thereby protecting* the iron from H+.
Linearly coordinated A bent Fe—CO The dioxygen ligand

CO binds ~ 25,000 geometry weakens binds to the iron-heme
more tightly to Fe the ligand interaction with an Fe-O-O angle
than dioxygen does. significantly. CO binds of ~ 115 – 159o.
only 200x stronger.
Discriminate in favor of O2 and against CO. How? *) heme iron oxidation is catalyzed by H+ that are reduced by the heme Fe
and that in turn reduce O2 to O2z−
The Electronic Structure of Hemoglobin/Myoglobin:
The Role of the Proximal (F8) α-subunit
Observation: Diamagnetic Spin Ground State for the Oxy-Form!
& Distal (E7) Histidine:
Fe-O-O difference between α and

β-chain in Hb is not significant
(within exp. error);
but very different from
Fe-O-O of 115o in Mb—O2
overlay
no H-bond
to His E7
β-subunit
Solution: Antiferromagnetic spin-spin coupling.
Dioxygen: Uptake, Transport & Storage

Status Quo
Hb binds dioxygen and transports O2 to the tissue.
Two strikingly similar proteins, hemoglobin (Hb) & myoglobin (Mb), How does Mb manages to get O2 transferred from Hb?
that immobilize and protect the active site, a heme—iron unit,
which reversibly bind dioxygen.
Hemoglobin is a much more
intricate and sentient molecule
But how does the system address the following important issues:
than Myoglobin is!
Hb binds dioxygen and transports O2 to the tissue. Hb transports H+ and CO2

How does Mb manages to get O2 transferred from in addition to O2
Hb—O2 + Mb to Mb—O2 + Hb?
O2 binding properties in Hb
Active tissue produces CO2 and H+. are regulated by interactions
Who takes care of the waste products? between separate, non-
adjacent sites.
Maternal blood needs to transfer O2 to fetal blood.
Hemoglobin is an
How do fetal red blood cells manage to receive O2? allosteric protein;
whereas Myoglobin is not!
5
Dioxygen binds cooperatively to hemoglobin!
The binding of O2 to hemoglobin enhances the binding of additional

O2 to the same hemoglobin (take advantage of high concentrations
of O2 in the lungs; sigmoidal curve ). Binding of O2 to Myoglobin is
not cooperative; hyperbolic cure).
Affinity of hemoglobin for O2 is pH dependent!
H+ and CO2 promote the release of bound dioxygen (for instance in

active tissues such as in muscles). Reciprocally, higher
concentrations of O2 promote the release of CO2 (e.g. in the lungs).
Dioxygen affinity of the tetrameric hemoglobin is regulated

Cooperative O2 Binding makes
by 2,3-BiPhosphoGlycerate (lowered by the presence of BPG)! Hemoglobin a More Efficient
Dioxygen Transporter
The Bohr Effect
H+ and CO2 Promote the Release of O2: The Bohr Effect (1904)
Most of the CO2 is transported as bicarbonate, which is formed within
red blood cells by the action of carbonic anhydrase:
CO2 + H2O Ù HCO3− + H+
The major portion of the Bohr Effect is due to the fact that
increasing p(CO2) causes a decreased red cell pH (acidosis).
A secondary part of the Bohr Effect is due to the fact that CO2
reacts covalently with hemoglobin to form carbamino-hemoglobin
which has a reduced O2 affinity.
R—NH2 + CO2 Ù R—NH—COO− + H+
The bound carbamates form salt bridges that stabilize the T-form!
Methanol (methyl alcohol) is highly poisonous because it is converted to a toxic product (formaldehyde) in a
reaction catalyzed by the enzyme alcohol dehydrogenase. Part of the medical treatment for methanol (The Tense-form of hemoglobin possesses a lower O2 affinity).
poisoning is to administer ethanol (ethyl alcohol) in large amounts. WHY???
Molecular Structure in the Region of the Heme:
Deoxyhemoglobin Oxyhemoglobin Hb/Mb is a very interrelated and complex system.
Controlling ligand binding: Distances in Å α-chain β-chain α-chain β-chain

Out-of-plane position of the
Hb is the a machine where atoms are moving parts.
Fe-Pheme 0.58(3) 0.50(3) 0.16(8) 0.00(8)
iron ion is not exclusively due Fe-PN 0.40(5) 0.36(5) 0.12(8) -0.11(8)
larger onic radius of the Fe(II). Movements in close proximity to the heme unit:
Fe-Nε(F8) 2.16(6) 2.09(6) 1.94(9) 2.07(9)
“It takes” less than 0.5 kcal to
place the Fe(II) ion in the Fe-Nporph(mean) 2.08(3) 2.05(3) 1.99(5) 1.96(6)
mean plane of the porphyrin. Nε-Nporph(mean) 3.26(4) 3.17(4) 2.9 2.8
Cδ-N3 porph 3.78(8) 3.74(8) 3.2(2) 3.1(2)
Cε-N1 porph 3.26(8) 3.12(8) 2.9(2) 3.0(2)
Angle between
Fe-Nε (heme ) 8(2) 5(2) 3 5
6
The binding of BPG to deoxy-Hb as viewed down the
The Movement of Fe(II) into the Heme molecule’s exact 2-fold axes (central cavity).
Plane triggers the T Æ R Conformation
Shift in the Quaternary Structure:
In the R-state, the central cavity is too

narrow to contain BPG
Expression of Hemoglobin Genes in Human Development:
The T Æ R transformation brings the two

b H helices together which narrows the
central cavity and expels the BPG. α-chain:
β-chain:
Adult hemoglobin: α2β2: Hb A
Fetal hemoglobin: α2γ2: Hb F
Dioxygen: Uptake, Transport & Storage

Why does BPG bind more weakly to fetal than to adult Hb?
(or: why is O2 transferred from maternal to fetal blood?)
Residue 143 in hemoglobin F is an uncharged serine instead of

the positively charged histidine in hemoglobin A

Heme Elek

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Dioxygen: Uptake, Transport & Storage:

M. F. Perutz et al. Acc. Chem. Res. (1987) 20, 309—321.

J. M. Riefkind Adv. Inorg. Biochem. (1988) 7, 155—241.

M. F. Perutz Annu. Rev. Physiol. (1990) 52, 1—25.

 R. E. Dickerson, I. Geis Hemoglobin 1983

D. Voet & J. G. Voet Biochemistry 2nd ed. 1995

L. Stryer Biochemistry 4th ed. 1997 050902

The Dioxygen-Carrying Team: Hemoglobin/Myoglobin

taken from: Dennis Kunkel Microscopy, Inc.

The richest source of

It is no surprise that the first x-

taken from: Kendrew at al. Nature (1960), 185, 422.

…requires an awful lot of imagination!

©2000 Howard Hughes Medical Institute

Front view: Side view:

Position Amino Acid Role

The imidazole ring of the distal His

Linearly coordinated A bent Fe—CO The dioxygen ligand

Fe-O-O difference between α and

Dioxygen: Uptake, Transport & Storage

 Hb binds dioxygen and transports O2 to the tissue. Hb transports H+ and CO2

 Dioxygen binds cooperatively to hemoglobin!

The binding of O2 to hemoglobin enhances the binding of additional

 Affinity of hemoglobin for O2 is pH dependent!

H+ and CO2 promote the release of bound dioxygen (for instance in

 Dioxygen affinity of the tetrameric hemoglobin is regulated

CO2 + H2O Ù HCO3− + H+

R—NH2 + CO2 Ù R—NH—COO− + H+

Controlling ligand binding: Distances in Å α-chain β-chain α-chain β-chain

In the R-state, the central cavity is too

The T Æ R transformation brings the two

Adult hemoglobin: α2β2: Hb A

Fetal hemoglobin: α2γ2: Hb F

Dioxygen: Uptake, Transport & Storage

Residue 143 in hemoglobin F is an uncharged serine instead of

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R. E. Dickerson, I. Geis Hemoglobin 1983

Hb binds dioxygen and transports O2 to the tissue. Hb transports H+ and CO2

Dioxygen binds cooperatively to hemoglobin!

Affinity of hemoglobin for O2 is pH dependent!

Dioxygen affinity of the tetrameric hemoglobin is regulated