Chemistry 5.

07SC Biological Chemistry I

Fall Semester, 2013

Lectures 4. Protein structure and function continued.

Quaternary Structure using Hb as an example and Mb for comparison. Many proteins are
composed of more than one polypeptide chain. You have already seen this with the collagen.
Quaternary structure can provide a basis for regulation at a distance: allosteric regulation and
cooperative behavior. We will see when studying metabolic pathways, that many of the
enzymes that control the flux through the pathway (rate-limiting step(s)) have quaternary
structures and exhibit cooperative behavior by binding small molecule metabolites. However,
there are many other proteins composed of multiple polypeptide chains where the chains act
independently.

I. Big Picture: Mb and Hb have different physiological roles. Mb is a monomer and is found in
tissues. It functions as a carrier of O2 to the mitochondrial in a cell. O2 has limited solubility (0.1
mM) and thus a carrier is required for rates of distribution to be sufficient for metabolism. O2 is
reduced to H20 in the respiratory chain in the mitochondria and the energy released is used to
make the energy currency of the cell, ATP.

Hb is a tetramer composed of two types of subunits designated α and β. These subunits are
structurally homologous to each other and to Mb (Figure 2). Hb is found in erythrocytes (red
blood cells (RBCs)) and also functions as an O2 carrier. It acquires O2 from air through the
lungs and delivers it through the circulatory system to the tissues where it is picked up by Mb.
The surface to volume ratio of vertebrate cells, in contrast with bacteria, often requires
transporters for small metabolites and Mb, for O2, is an example. The RBCs also retrieve CO2
from the tissues, the end product of oxidative metabolism, and deliver it to the lungs where it is
exhaled (Figure 1). The RBCs deliver CO2 as part of the H+/HCO3- equilibrium (think about
Problem Set 1) and via Hb itself which is able to form carbamates (-NHCO2-) via reaction of the
lysines on its surface with CO2. These carbamates are acid labile and decompose to CO2 and

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non-modified Hb. The O2 binding properties of Mb and Hb are distinct and dependent on
quaternary structure. The distinctive bind properties (hyperbolic versus sigmoidal) are essential
for their physiological function (see Figure 4).

Figure 1. Cartoon of O2 transport in vertebrates. Hb transports O2 to the tissues (muscle). Metabolism in

muscle, such as glucose oxidation, results in CO2 production and NADH formation. NADH oxidation is
coupled to reduction of O2 in the mitochondrial respiratory chain. The energy released from H2O
formation, generates a proton gradient that can be used for ATP production. Mb transports O2 to the
mitochondria for this purpose. The resulting CO2, the end product in metabolism, is returned to the lungs
via the blood where it is exhaled.

Structures: Mb and Hb (Figure 2). Note in the case of Hb that there are multiple subunit
interactions: α2β2, α1β1 and β2α1 and α2β1. The movement between subunits occurs
predominantly through the second set of subunit interfaces.

For additional information, please see the Molecule of the Month article on Hemoglobin from
the RCSB PDB.

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A. B.

Figure by O'Reilly Science Art for MIT OpenCourseWare.

A. PDB: 3RGK
Hubbard, Stevan R., Wayne A. Hendrickson, David G. Lambright, and Steven G. Boxer. "X-ray crystal structure of a
recombinant human myoglobin mutant at 2· 8 Å resolution." Journal of molecular biology 213, no. 2 (1990): 215-218.
B. PDB: 2HHB.
Fermi, G., M. F. Perutz, B. Shaanan, and R. Fourme. "The crystal structure of human deoxyhaemoglobin at 1.74 Å
resolution." Journal of molecular biology 175, no. 2 (1984): 159-174.

Figure 2. Crystal structures of myoglobin and hemoglobin. Heme molecule bound is shown in grey
space-filling balls. A. Structure of the monomeric myoglobin. B. Structure of tetrameric hemoglobin,
showing similarity with myoglobin of the β1 subunit. Red and pink monomers are alpha. Blue and grey
monomers are beta.

II. Myoglobin:

A. See the quaternary structures above. Both Mb and Hb reversibly bind to O2 via Fe2+ in
protoporphyrin IX (heme, see Figure 3), a key cofactor (see Lexicon). An octahedral complex is
formed in which the nitrogens of the pyrrole rings are equatorial ligands and the O2 and a His (in
the F helix) are the axial ligands. The protein structure prevents oxidation of Fe2+ to Fe3+ most of
the time. If oxidation does occur, there is a protein called Cytb5 (a heme protein) that reduces
Fe3+ to Fe2+.

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A. B.

Figure 3B from S. Dutta and D. Goodsell. (May 2003) The RCSB PDB "Molecule of the Month": Hemoglobin.
doi:http://dx.doi.org/10.2210/rcsb_pdb/mom_2003_5.

Figure 3. Protoporphyrin IX. A. Protoporphyrin IX with a coordinated Fe2+ atom. B. Deoxy Hb (left)
and oxy Hb (right) forms, with movement of the iron, porphyrin ring and His upon O2 binding.

B. O2 binding to Mb can be measured experimentally and the results of a typical experiment are
shown in the graph below. The rectangular hyperbola describes O2 binding to Mb. The behavior
of Mb (red line) is distinct from that described for Hb (blue and green lines).

Figure by O'Reilly Science Art for MIT OpenCourseWare.

Figure 4. O2 dissociation curves for Mb and Hb in whole blood and their differences. Mb’s O2
dissociation curve is hyperbolic: Its p50 (the value of pO2 when YO2 = 0.5) is 2.8 torr where 1 torr = 1
mm Hg at 0ºC; 760 torr = 1 atm). Hb exhibits a sigmoidal binding curve and the amount of O2 bound at
100 torr (arterial pressure) is substantially different from that at venous pressure (20-30 torr) where the O2
needs to be unloaded.
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C. Mb binding to O2 is described by a standard binding analysis:
Mb + O2  Mb•O2 and the
Kd = [Mb][O2]/[Mb•O2] and YO2 = Mb•O2/ [Mb•O2 + Mb]
The binding is described as the fraction of Mb saturated by O2 that is,
(YO2) = [O2]/ (Kd + [O2]).
Since O2 is a gas, its concentration is expressed as partial pressure of O2 (pO2). p50 is the partial
pressure of O2 when 50% is bound.

III. Hemoglobin
Hemoglobin has four subunits and the difference in its ability to bind O2 relative to Mb is based
on the communications between the subunits.

Historical Digression
Max Perutz (1914 – 2002) began his work on Hb in 1937, and Hb was the first protein
crystallized (1949). It was nearly fifteen years later when the x-ray structure was solved (1962)
and it required Perutz to invent the method of isomorphous replacement to solve the phase
problem. Hb was a first in many categories, and its rich history is fascinating. Vernon Ingram
(MIT Biology) established for the first time that a “single” mutation of an amino acid (E to V) is
responsible for a genetic disease – sickle cell anaemia. It was Linus Pauling (again!) who
proposed this theory first. Hb was the first system, and still today is the best studied system, in
which cooperativity of ligand binding was established.
End historical digression

Binding of one ligand effects the binding of additional ligands. Based on the observation of
cooperativity of binding, a number of theories (Monod/Wyman/Changeux; Koshland – covered
more below) and a phenomenological description were put forth to explain the experimental
observations. These theories describe a major mechanism of regulatory control for key (rate-
limiting) enzymes in metabolic pathways.

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We will focus on two issues (expanded on below):
A. the basis for the sigmoidal O2 binding curve of Hb (Figure 4).
B. the mechanism of allosteric regulation by H+ (the Bohr effect) or 2, 3-
bisphosphoglyceric acid (BPG – see structure below).

A. Cooperativity in O2 binding to Hb is displayed in the graph above. One can observe that in
the lungs where the pO2 is 100 torr, that Hb is >90% saturated with O2. In the tissue however,
where pO2 is 20 to 30 torr, the Hb binds O2 at 50% saturation. Under the same conditions, the
graph also shows that O2 is tightly bound to Mb. Thus Mb is able to bind the O2 released from
the Hb.
New vocabulary used in the description of cooperative binding of ligands.
Allostery = binding of a ligand to a specific site, affects binding of the same ligand or a
different ligand to an additional site.
Homotropic = both ligands are the same. In the case of Hb, the ligand is O2.
Heterotropic = the ligands are not the same. O2 binds and the allosteric effectors can be
H+ (Bohr effect), Cl-, CO2, bis-phosphoglyceric acid, BPG.
There are a number of models that describe cooperative behavior: In the case of hemoglobin, at
issue is how the binding of O2 of one subunit can increase the binding of O2 to additional
subunits. The distances between the hemes in the subunits are 35 and 27Å!

Monod/Changeux/Wyman model (1) and Koshland/Nemethy/Filmore model (2) to explain

cooperativity. While these models are mathematically distinct, both intuitively describe the
cooperative behavior and in most cases, it is experimentally difficult to distinguish between
them.

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1. In the Monod model described in Figure 5A, T is the tight, or taut, state (by convention) that
weakly binds O2, while R is the relaxed state that tightly binds O2.
A. B.

Figure by O'Reilly Science Art for MIT OpenCourseWare.

Figure 5. Monod/Changeux/Wyman model.

Assumptions of the Monod model:

a. The protein must exist in at least two conformations that are in equilibrium: the R and
T states. T is the state where substrate has low affinity for Hb, while R has high
affinity.

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 b. The states must have multiple equivalent binding sites, quaternary structure. There
are 4 in the case of Hb.
c. The protein is either in the all R or all T states. There are NO mixed states.

2. Koshland/Nemethy/Filmore KNF sequential model (Figure 6).

Figure by O'Reilly Science Art for MIT OpenCourseWare.

Figure 6. Koshland/Nemethy/Filmore model.

Assumptions of the Koshland model (2):

a. The binding affinity to one site influences the binding affinity to a neighboring site.
b. This model is able to account for negative cooperativity (binding at one site, decreases
the affinity at the second site) that is seen in a number of enzymatic reactions.

Neither model in its pure form accounts for the experimentally observed behavior of Hb. A
combined model is required.

We have 100s of structures from different species of Hb in the oxygenated and deoxygenated
states. The molecular basis for interconversion of the R and T states is understood based on
structures and decades of study. The conformational change is triggered by binding of O2 to one
heme of Hb (Figure 7). Blue is deoxyHb and red is oxyHb. In the deoxyHb, Fe2+ is too large to
fit into the opening in the porphyrin ring. Upon binding of O2 to Fe2+, the size is reduced and
Fe2+ fits into the hole, dragging down the His (Figure 3B) axial ligand (F8) and the F helix along
with it as it comes into the plane of the porphyrin ring.

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© John Wiley & Sons. All rights reserved. This content is excluded from our Creative Commons license. For more information,
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Source: Voet, Donald, and Judith G. Voet. "Biochemistry." John Wiley & Sons, 2010.

Figure 7. Conformational changes occur in both the F helix of hemoglobin and the heme upon oxygen
binding. When oxygen is bound, the protein transitions from the deoxygenated structure (blue) to the
oxygenated structure (pink) as the F helix moves down, pulled by its heme-coordinating His F8.

B. Regulation by H+ or 2, 3-bisphosphoglyceric acid (BPG). We will focus briefly on the Bohr

effect. The diagram below shows the effect of pH on O2 binding to Hb. The experimental
observation is that at pH 7.2 and 20 torr, more O2 is unloaded than at pH 7.6 (Figure 8). Can we
provide an explanation for this observation based on structures?

Figure by O'Reilly Science Art for MIT OpenCourseWare.

Figure 8. Binding of oxygen to the heme of hemoglobin is pH dependent.

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To think about the basis of this pH effect you need to recall PS 1 and the CO2/HCO3- equilibria.
You also need to know that deoxyHb is a stronger base than O2•Hb. Finally, in RBCs, there is
an enzyme carbonic anhydrase, which catalyzes the hydration of CO2 by water.

and O2Hb + H+  [H+]DeoxyHb + O2

Using these equations you can think about how to maximize unloading of O2 in the tissues and
unloading of CO2 in the lungs.

First example: In muscle during exercise, glucose is converted to CO2. The CO2 then diffuses to
the capillaries and RBCs and equilibrates to H+ and HCO3-. Some of the side chains of Hb act as
a buffer. The H+s bind to deoxyHb and thus shift the equilibrium (above graph, right) to the
right, releasing more O2.

Second example: The deoxyHb is transported through the circulatory system back to the lungs
where it picks up O2. O2 shifts the equilibrium to the left generating H+. The protons then react
with HCO3- to generate CO2 and H2O. The CO2 is exhaled. In addition, the H+ (transient
reduction in pH) can react with the carbamylated-Hb to also release CO2.

Medical Digression - Mutant Hemoglobins

Many mutations in Hb are associated with disease – often anaemia – a lower number of red
blood cells and not enough Hb. The most common disease is sickle cell anaemia. In the US,
there are 70 000 people affected by sickle cell anaemia, though about 2 million are heterologous
for the sickle cell trait. Its occurrence is roughly 1 in 36 000 Hispanic Americans and 1 in 500
African Americans. The disease is recessive, requiring two copies of the sickle cell gene
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containing the single mutation and is characterized by the C shaped erythrocytes that tend to get
stuck in blood vessels, form clots causing damage to potentially many organs (bone pain,
necrosis, skin ulcers), and are very fragile compared to wild type (normal) erythrocytes. The
lifetime of sickle cell erythrocytes is about a tenth the length of the wild type cells (10-20 days
instead of ~120 days). It is this sickle cell trait that resistance to malaria parasites persists. For
more information on sickle cell anaemia, see the webpage on Sickle Cell Diseases at the National
Heart, Lung, and Blood Institute from the National Institute of Health.
End medical digression

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5.07SC Biological Chemistry I

Fall 2013

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