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May 2003: Hemoglobin
Red Blood, Blue Blood
Ever wondered why blood vessels appear blue? Oxygenated blood is bright red:
when you are cut, the blood you see is brilliant red oxygenated blood.
Deoxygenated blood is deep purple: when you donate blood or give a blood
sample at the doctor's office, it is drawn into a storage tube away from oxygen,
so you can see this dark purple color. However, deep purple deoxygenated
blood appears blue as it flows through our veins, especially in people with fair
skin. This is due to the way that different colors of light travel through skin:
blue light is reflected in the surface layers of the skin, whereas red light
penetrates more deeply. The dark blood in the vein absorbs most of this red
light (as well as any blue light that makes it in that far), so what we see is the
blue light that is reflected at the skin's surface. Some organisms like snails and
crabs, on the other hand, use copper to transport oxygen, so they truly have
blue blood.
Hemoglobin is the protein that makes
blood red. It is composed of four
protein chains, two alpha chains and
two beta chains, each with a ring-like
heme group containing an iron atom.
Oxygen binds reversibly to these iron
atoms and is transported through
blood. Each of the protein chains is
similar in structure to myoglobin
(presented in the January 2000
Molecule of the Month), the protein
used to store oxygen in muscles and
other tissues. However, the four
chains of hemoglobin give it some
extra advantages.
Use and Abuse of Hemoglobin
Aside from oxygen transport, hemoglobin can bind and transport other
molecules like nitric oxide and carbon monoxide. Nitric oxide affects the walls
of blood vessels, causing them to relax. This in turn reduces the blood
pressure. Recent studies have shown that nitric oxide can bind to specific
cysteine residues in hemoglobin and also to the irons in the heme groups, as
shown in PDB entry 1buw. Thus, hemoglobin contributes to the regulation of
blood pressure by distributing nitric oxide through blood. Carbon monoxide,
on the other hand, is a toxic gas. It readily replaces oxygen at the heme
groups, as seen in PDB entry 2hco and many others, forming stable complexes
that are difficult to remove. This abuse of the heme groups blocks normal
oxygen binding and transport, suffocating the surrounding cells.
May 2003: Hemoglobin
Artificial Blood
Blood transfusions have saved countless lives. However, the need for matching
blood type, the short life of stored blood, and the possibility of contamination
are still major concerns. An understanding of how hemoglobin works, based on
decades of biochemical study and many crystallographic structures, has
prompted a search for blood substitutes and artificial blood. The most obvious
approach is to use a solution of pure hemoglobin to replace lost blood. The
main challenge is keeping the four protein chains of hemoglobin together. In
the absence of the protective casing of the red blood cell, the four chains
rapidly fall apart.
To avoid this problem, novel hemoglobin
molecules have been designed where two
of the four chains are physically linked
together, as shown in PDB entry 1c7d. In
that structure, two additional glycine
residues form a link between two of the
chains, preventing their separation in
solution.
Hemoglobin Cousins
Looking through the PDB, you will find many different hemoglobin molecules.
You can find Max Perutz's groundbreaking structure of horse hemoglobin in
entry 2dhb, shown in the picture here. There are structures of human
hemoglobins, both adult and fetal. You can also find unusual hemoglobins like
leghemoglobin, which is found in legumes. It is thought to protect the oxygensensitive bacteria that fix nitrogen in leguminous plant roots. In the past few
years some hemoglobin cousins called the "truncated hemoglobins" have been
identified, such as the hemoglobin in PDB entry 1idr, which have several
portions of the classic structure edited out. The only feature that is absolutely
conserved in this subfamily of proteins is the histidine amino acid that binds to
the heme iron.
May 2003: Hemoglobin
Cooperation Makes It Easier
Hemoglobin is a remarkable molecular machine that uses motion and small
structural changes to regulate its action. Oxygen binding at the four heme sites
in hemoglobin does not happen simultaneously. Once the first heme binds
oxygen, it introduces small changes in the structure of the corresponding
protein chain. These changes nudge the neighboring chains into a different
shape, making them bind oxygen more easily. Thus, it is difficult to add the
first oxygen molecule, but binding the second, third and fourth oxygen
molecules gets progressively easier and easier. This provides a great advantage
in hemoglobin function. When blood is in the lungs, where oxygen is plentiful,
oxygen easily binds to the first subunit and then quickly fills up the remaining
ones. Then, as blood circulates through the body, the oxygen level drops while
that of carbon dioxide increases. In this environment, hemoglobin releases its
bound oxygen. As soon as the first oxygen molecule drops off, the protein
starts changing its shape. This prompts the remaining three oxygens to be
quickly released. In this way, hemoglobin picks up the largest possible load of
oxygen in the lungs, and delivers all of it where and when needed.
In this figure, the heme group of one subunit is shown in the little circular
window. The oxygen molecule is shown in blue green. As it binds to the iron
atom in the center of the heme, it pulls a histidine amino acid upwards on the
bottom side of the heme. This shifts the position of an entire alpha helix,
shown here in orange below the heme. This motion is propagated throughout
the protein chain and on to the other chains, ultimately causing a large rocking
motion of the two subunits shown in blue. The two structures shown are PDB
entries 2hhb and 1hho.
May 2003: Hemoglobin
Troubled Hemoglobins
The genes for the protein chains of
hemoglobin show small differences
within different human populations, so
the amino acid sequence of
hemoglobin is slightly different from
person to person. In most cases the
changes do not affect protein function
and are often not even noticed.
However, in some cases these
different amino acids lead to major
structural changes. One such example
is that of the sickle cell hemoglobin,
where glutamate 6 in the beta chain is
mutated to valine. This change allows
the deoxygenated form of the
hemoglobin to stick to each other, as
seen in PDB entry 2hbs, producing
stiff fibers of hemoglobin inside red
blood cells. This in turn deforms the
red blood cell, which is normally a
smooth disk shape, into a C or sickle
shape. The distorted cells are fragile
and often rupture, leading to loss of
hemoglobin. This may seem like a
uniformly terrible thing, but in one
circumstance, it is actually an
advantage. The parasites that cause
the tropical disease malaria, which
spend part of their life cycle inside red
blood cells, cannot live in the fiberfilled sickle cells. Thus people with
sickle cell hemoglobin are somewhat
resistant to malaria.
Other circumstances leading to troubled hemoglobins arise from a mismatch in
the production of the alpha and beta proteins. The structure requires equal
production of both proteins. If one of these proteins is missing, it leads to
conditions called Thalassemia.
May 2003: Hemoglobin
Exploring the Structure
You can look at the binding of oxygen up close in two structures of human
hemoglobin. PDB entry 2hhb shows hemoglobin with no oxygen bound. In this
picture, the heme is seen edge-on with the iron atom colored in gold. You can
see the key histidine reaching up on the bottom side to bind to the iron atom.
In PDB entry 1hho, oxygen has bound to the iron, pulling it upwards. This in
turn, pulls on the histidine below, which then shifts the location of the entire
protein chain. These changes are transmitted throughout the protein,
ultimately causing the big shift in shape that changes the binding strength of
the neighboring sites.