Transcript Hemoglobin and Cytochrome c

The relationship between structure and
function is a central concept in
Biochemistry. The book emphasizes
myoglobin and hemoglobin to illustrate
the ideas
These proteins bind and release O 2. Hb
also removes CO 2. .
Mb - single polypeptide chain folded
about a prosthetic group “heme”. Hb is a
tetramer of the form α 2β 2 with two
equivalent subunits.
Mb: 153 amino acids MW ~ 17kD
Secondary structure 77% α-- helix, 23%
random coil. 121 residues in helical form
with lengths running from 7  26
residues.
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Helical segments A  H fold back on
each other to form a globular cavity
for the heme group. Proteins without
prosthetic groups cannot bind O 2 .
But transition metals such as iron (Fe
II) and Copper (Cu II) can bind O 2 .
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Myoglobin
Heme
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Hemoglobin (Hb) functions to transport oxygen in the
blood of all warm-blooded animals. It is a multimeric
protein consisting of
four subunits, alpha1 , beta1, alpha2 and beta2. All four
subunits resemble both themselves and myoglobin. The
subunits are symmetrically arranged. Note the
extensive contacts at between alpha and beta subunits,
while there is little alpha-alpha and beta-beta contact
(as they face each other across a 20 Å channel that runs
through the center of the protein.) Also note the heme
group located within a deep cleft in the side of each of
the subunits. The heme groups are both the binding site
for oxygen and the starting point for the mechanism of
oxygen binding cooperativity. Above is a diagram of
the alpha1 subunit of Hb. Oxygen binds to the Fe(II)
atom, directly below the plane of the heme group and
opposite the proximal histidine. Note how the top and
bottom of the heme group are surrounded by the
protein globule. This prevents two Hb heme groups
from binding to the same oxygen molecule, which
would allow one heme group to catalyze the
autooxidation Fe(II) to Fe(III) in the other, resulting in
irreversable oxygen binding.
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Hemoglobin: Structure & Function
Hemoglobin is a large protein (66.7 kD)
coupled to four porphyrins or heme moities.The
globin portion of hemoglobin consists of four
polypeptide chains ( “a” with 141aa and “ß”
with 146aa )arranged in pairs forming a
tetramer. Each globin chain is covalently
attached to a heme moiety. The bonds between
a and ß chains are weaker than between similar
globin chains, forming a natural cleavage plane,
the a1ß2 interface, important for oxygen
binding and release.
When this cleavage is open [R (relaxed)
state] oxygen can bind (high oxygen affinity).
When the two a1ß2 interfaces are closely bound
[T (taut) state] the Hgb molecule has a low
affinity for oxygen.
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The globin fold is found in many related
proteins such as myoglobin and hemoglobin.
Usually, it has eight a-helices, designated AH. These eight helices pack into two layers,
making an angle of about 50o between them.
In each layer, these a-helices are arranged in
antiparallel fashion into a pattern called a
Greek key helix bundle. Specifically, the
sperm whale myoglobin has 153 aa residues
and a heme group. The eight helices in
myoglobin have the following aa segments:
Helix
A: SER3 -- GLU18
B: ASP20 -- SER35
C: HIS36 -- LYS42
D: THR51 -- ALA57
E: SER58 -- LYS77
F: LEU86 -- THR95
G: PRO100 -- ARG118
H: LY124 -- LEU149
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The heme (Fe(II)-protoporphyrin IX) is
the prosthetic group of myoglobin as
well as hemoglobin; it is the oxygen
binding site. Structurally, the heme is an
iron-coordinated porphyrin, a macro
cyclic ring system composed of four
pyrroles with four methyl, two vinyl,
and two propionic acid side chains. All
atoms in the tetrapyrrole ring (neglecting
the side chains) lie nearly in the same
plane.
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The Fe atom is chelated by a conjugated
tetrapyrrole ring system - Protoporphyrin IX.
The protoporphyrin IX-Fe II complex is called
heme.The visible absorption spectrum of heme is
responsible for the color of blood. Heme is concovalently bound to globin in a hydrophobic
crevice in both Mb and Hb.
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Each heme group can bind one oxygen
molecule. Thus, the hemoglobin tetramer can
bind a total of four oxygen molecules.
Hemoglobin also serves the purpose of
transporting carbon dioxide. Oxygen is bound
by hemoglobin at the lungs, carried through
the blood stream to various tissues, where it
gives up the oxygen, and binds carbon dioxide
and transports it back to the lungs. Carbon
dioxide is bound as the bicarbonate ion,
HCO3-, at the dimer interface and not to the
heme.
The binding of oxygen to hemoglobin is
cooperative. Thus, binding of one oxygen
causes the next oxygen to bind more strongly,
the third oxygen even more strongly and so
on. In fact, hemoglobin is usually observed in
only two states - all four chains carrying
oxygen, or completely oxygen free.
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Binding of Oxygen
Fe II normally has ligands around it. The first 4 are
occupied by binding to the four nitrogen atoms of the
heme. This binding leaves 2 binding sites  ring. In
Mb one site is the N atom of His 93.
Residue 93 is in the F helix (Called His F8 or
proximal His)
In Deoxy Mb, the remaining site is H 2 O.
When it binds O 2 occupies this site. On the other side
of the bound O 2 ,lies His 64 (E7) called the distal His
Therefore O 2 is sandwiched between the heme Fe
and the ring N of the distal His. There is similar
binding in Hb.
THE HYDROPHOBIC EN VIRON MENT IN THE
PROTEIN SER VES TO PRE VENT THE
OXIDATION OF Fe IIFe III
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Changes in the heme group upon O2 binding to the  chains
of hemoglobin shown in this slide and the previous one,
The heme groups of the Deoxy (T) and oxy (R) structures are shown.
The two oxygen atoms of the bound O2 are shown below the heme group.
Upon binding, the iron atom moves about 0.6  from above into the
plane of the heme.
This action pulls with it His 87, also known as F8, the 8th residue of
the F helix, and the F helix of which it is a part. This in turn produces
changes in the FG corner on the left, which is in contact with the
other chain across the 12 interface. It is this interface which
changes during the T R quaternary structure change. The above
mechanism describes a plausible, but unproven sequence of events
by which oxygen binding affect the quaternary structure. The dashed
lines are H-bonds , presumably involved in orienting this His F8
side chain.
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Hemoglobin can exist in two different
conformations: The T-form and the R-form. The Tform is characterized by the presence of many salt
linkages (or bridges) between different subunits,
and these linkages are broken as hemoglobin
undergoes the T --> R transition upon oxygenation.
For instance, two pairs of salt bridges are present in
deoxyhemoglobin, involving the C-terminal groups
(Arg141) of the a-chain at the a1/a2 interface.
Specifically, the negatively charged carboxyl group
of Arg141 of each a-subunit makes a salt bridge
with the positively charged amino group of Lys127
of the other a-subunit. And the positively charged
guanidino group of Arg141 interacts
electrostatically with the carboxyl group of
Asp126's side chain.
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A plot of the the 12 interface in the R and T
states of hemoglobin as indicated , illustrating the
large differences. At the top is the C helix 1
chain, at the bottom the irregular corner linking
the F and G helices of the 2 chain. The
conformations of both are virtually the same in
both the R and T structures, but the contacts differ
markedly owing to a shift of one subunit relative
to the other. For example His 97 of the  chain
is in contact with thr 41 of the  chain in T, but
with thr 38, one turn back along the C helix in R.
Intermediate positions would be unstable since
his 97 and thr 41 would be too close together.
Therefore, molecules must either be in the T or R
states, irrespective of the number of bound
ligands
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F Helix Transition
Recall that there is extensive contact between the alpha-beta
interfaces in Hb. While the alpha1-beta1 and alpha2-beta2
interfaces are rigid, the alpha1-beta2 and alpha2-beta1
interfaces are somewhat flexible. This flexibility allows the
translation of the F helix to result in a quaternary shift at the
alpha1-beta2 and alpha2-beta1 interfaces. Shown above, the
beta2 F helix terminus- His 97- moves down one turn of the
alpha1 C helix, from in between the Pro 44 and Thr 41
residues to in between the Thr 41 and Thr 38 residues. No
intermediate position would be stable- the His 97 and Thr 41
residues would bump into each other. This accounts for the
absence of any stable intermediate form between the T and R
states. Accompanying the F helix translation is extensive
breaking and reforming of salt bridges and H- bonds,
Below is a view of a portion of the alpha1-beta2 interface. It
is possible to see the chain reaction of events that lead to the
reorientation at the alpha1-beta2 (and alpha2-beta1)
interfaces. Upon oxygen binding (not shown), the heme's
doming is flattened, pulling the proximal his (here His 92)
0.6 Å towards the heme. This forces the attached F helix to
undergo a 1 Å translation along the heme plane, pulling with
it the attached FG helix. This movement results in the
reorientation at the alpha1-beta2 interface, where the end of
the F helix and the side of the FG helix are forced to break
and reform many hydrogen bonds and salt bridges (The
hydrogen bond between Tyr 42 and Asp 99 is one example).
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The binding of oxygen rotates the
globin chains, moving the ß chains
together and sliding the a1ß2
interfaces apart (the R state) thus
increasing the oxygen affinity of Hgb.
Hemoglobin must bind O2 at high O2
tension and release it at low O2
tension. With deoxygenation the a1ß2
interface tightens lessening the affinity
of Hgb for oxygen. This conformation
is stabilized by proton binding and
2,3-DPG.
Decreasing pH strengthens the a1ß2
interface, stabilizing the low-affinity
(T) conformation and releasing O2 .
This is the Bohr effect.
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Away from the cell Fe II slowly oxidizes to
become met Mb-Met proteins don’t bind O 2 .
When O 2 binds, a temporary rearrangement of
electrons occurs. When the O 2 is released, the Fe
remains Fe II and can bind O 2 once again.
To understand the function (binding and release
of O 2 ),we some elementary Physical Chemistry
[O 2] in fluid  P
Law
O2
(gas). This is called Henry’s
So that [O 2] is expressed as the pressure of O 2.
It is easy to measure the fraction of binding sites
occupied on the protein from changes in the
visible absorption spectrum when the binding
sites are occupied.
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The Significance of the Sigmodial Oxygen Binding
Curve
The significance of the sigmodial O2 saturation curve of Hb
can be appreciated from the graph below. While both Mb and
Hb will be saturated with O2 at the partial pressure of O2 in
the lungs, only hemoglobin will release significant amounts
of O2 at the partial pressure of O2 present in the tissues. In
fact, the O2 released by Hb can then be taken up by Mb for
O2 storage in those tissues, such as muscle, that have
significant amounts of Mb.
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The different behavior of oxygen binding of
myoglobin and hemoglobin can be summarized as
follows. Myoglobin binds O2 under conditions
where hemoglobin releases it. This is below 20
Torr in muscle tissue. At this pressure,
hemoglobin releases almost all of its oxygen and
myoglobin binds the freed oxygen at over 90%.
The hyperbolic curve of Mb binding is typical for
non-cooperative processes, while the sigmoidal
curve of Hb binding is typical for cooperativity
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Summary of binding methods for one site, one step, binding
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Some common methods of plotting binding data,
using theoretical curves constructed for the
simple reaction
P + A  PA
A) Normal hyperbolic relationship between
binding and free ligand concentration.
B) A logarithmic scale demonstrating the large
range of ligand concentrations required for a
complete binding curve.
C) A Scatchard plot-The negative slope gives the
value of the association constant.
D) A Hill plot. Both the liganded and free protein
concentrations are required for this plot. The plot
is used primarily for studies of cooperative
binding.
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A. Allosterics and conformational changes
Conformational changes in proteins
(alterations in the typically in the tertiary
/quarternary structure) are critical to their
function and regulation. Changes in protein
conformation facilitate movement,
catalysis, release of molecules (oxygen),
binding, etc.
Since conformational changes are both
the result of and lead to alterations in noncovalent interactions within and between
protein(s), regions in proteins must be able
to allow the structural changes (breaking
and forming new non-covalent
interactions).
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Conformational changes which result from
an interaction with a specific small
molecules are often described as
Allosterics.
Allosteric interactions (from the Greek
allos, other) = interactions or changes in
conformation (shape) of a regions in a
protein (or another protein) which
INDUCES a change in shape in another
part of the same molecule (or another
molecule)
Allosteric interactions typically occur
when a specific small molecule, called an
allosteric modulator or allosteric effector,
binds to a protein (often an enzyme) and
modulates its activity. The allosteric
modulator typically binds reversibly at a
site often separate from the functional
binding site of the protein.
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thereby
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Most recent results on structure changes . How
are the tertiary and quaternary structure
changes related to each other ?
The truth apparently lies somewhere between the
MWC model and the Koshland model. The tertiary
structure changes that accompany oxygen binding
can be tolerated up to a certain point before the T
 R switch. Apparently whenever one site is
occupied on each of the two  dimers, the
whole molecule adopts the R quaternary structure.
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The red blood cell contains high levels of bisphosphoglycerate
(BPG), which is an ALLOSTERIC EFFECTOR of Hb's affinity
for O2. BPG binds strongly to the deoxy form of Hb, but only
weakly to the oxy form. Thus, BPG favors the deoxy
conformation (bpg). The importance of bisphosphoglycerate
(BPG) as an allosteric regulator of hemoglobin's O2 affinity
High altitude adaptation
Adaptation to high altitude is a complex physiological process
that involves many events. One event that occurs within 24
hours is an increase in the content of BPG in the erythrocyte.
The effect of the increased concentration of BPG is to reduce
the affininity of hemoglobin for O2, which increases the
efficiency of O2 delivery to tissues
The Physiological Transport of O2 and CO2
How do all these allosteric effects combine to allow Hb to carry O2
from the lungs (gills) to the tissues and to carry CO2 from
the tissues to the lungs (gills)?
As shown below, Hb stripped of all its allosteric effectors has to
high an affinity for O2 to allow effective transport of O2 to tissues.
However, the presence of both CO2 in the tissues and BPG in
the red blood cell create a situation in which O2 is efficiently
transported from lung to tissue.
We can summarize these events as follows:
1.In the lungs the partial pressure of O2 is high, which
overcomes any negative allosteric effects and causes
complete oxygenation of Hb.
2.As Hb-O2 enters the tissues the presence of CO2 and a
lowered pH combine to favor the deoxy conformation and
release of O2.
3.The presence of BPG aids the delivery of O2 by
favoring the deoxy conformation.
4.Deoxy Hb binds CO2.
5.The deoxy Hb returns to the lungs where the pH is
higher, the O2 content higher and the CO2 content is lower.
All these factors favor reverse of the carbamation reaction,
deprotonation of His 146 and the formation of oxy Hb.
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Genes present today are the product of evolutionary divergence.
Proteins then accumulate mutations unless natural selection has
acted.
Overall mutation rates seem to occur at a rate of 1/100 (million years).
Neutral mutation rates are a fraction of this and vary with each
nucleotide and each gene. Mutations that adversely affect the gene
are (presumably) selected against. The number of mutations that
may be tolerated varies among genes.
Cytochrome C
Part of the chain of linked oxidations and reductions involved with
biological electron transport. Sequences from many organisms
are available.
The figure below shows a difference matrix giving the number of
amino acid differences in cytochrome C among a few selected
eukaryotic species.
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A composite of sequences , where every amino acid that has been found
at every position is listed below. 41 species are included . The numbering system used is that of the mammalian proteins, which start at 1
and end at 104. The longest proteins included in the Table start at -8 and
end at 105.
Wide variation occurs at 60, 89,92. No variation is found at other
positions such as 14,17, to which the heme group in this protein is
attached covalently. Positions 76-80 are also invariant
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The evolutionary process of divergence may be tracked by
reconstructing a phylogenetic tree from the amino acid changes.
This is a computer based calculation, since 2 million trees are
possible with 10 species.
Phylogenetic tree constructed from the sequences of cytochrome C. The
numbers of amino acid changes along each segment are listed.
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Mutations implied by Divergence
The non-random nature of the pairs of
amino acids that replace each other in a number
of different proteins are shown in the matrix
below.
The most prevalent replacements occur in amino
acids with chemically similar amino acid side
chains:
Gly/Ala, Ala/Ser, Ser/Thr, Ile/Val/Leu , Lys/Arg ,
Tyr/Phe
This bias in amino acid replacements does not
arise from the nature of the genetic code, even
though amino acids differing by single
nucleotides in their codons are most likely to
replace each other.
The second nucleotide in the codon is the most
important in specifying the chemical nature of the
amino acid; changes in the first interconvert
similar amino acids, changes in the third usually
produce no change in amino acid.
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The expected distribution with random basepair changes and random use of the code is
shown in the upper half of the Figure on the next
page. This distribution is quite different from
that actually observed The relative mutabilites
of residues during evolution is shown in the
Table below.
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Rates of Divergence in Evolution
The specific rates at which various proteins
have evolved are shown below for
fibrinopeptides A and B, cytochrome C
and hemoglobin. For each protein the rate is a
constant which differs from the others.
How do we explain the wide range of
evolutionary rates among proteins and the
nearly constant rate in time for the individual
proteins ?
Idea: If the precise amino acid sequence was
not critical for protein function, then a large
fraction of the total mutations would be
neutral, and the protein would evolve quite
rapidly.
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e.g. The fibrinopeptides seem to only block the
aggregation of fibrinogen. At the other end of the
spectrum, a protein for which very few amino
acid rates are acceptable would have a very slow
rate of evolution.
The example is cytochrome C , which must
interact with several proteins in its biological
function of transferring electrons.
The neutral mutation rate would be different for
each position within the gene or protein. The third
nucleotide base pair in many codons is not
specific for the amino acid coded and is
found to have evolved most rapidly.
In contrast, some amino acids with vital roles
can’t be changed, as we previously noted.
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Variants in Hemoglobin
Human cells carry two copies of each
chromosome. If there is a normal () and
mutated (*) form of the  chain gene
in Hb, an individual can have three possible
combinations of these genes, as follows:
(I)  homozygous (normal)
(II)* heterozygous (mixed)
(III) ** homozygous variant.
Individuals with  will produce normal
 Hb chains * produces both
normal and variant chains while, **
produces only variant chains.
300 Hb variants are known
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Sickle cell disease Glu  val at residue 6 of the
 chain causes deoxy Hb to polymerize,
thereby producing the characteristic sickling.
This leads to blockage of capillaries carrying
blodd and a sickling crisis.
Interesting issue:
Why does this apparently detrimental variant
persist ?
-only homozygotes suffer from sickle cell
anemia
-heterozygotes don’t. 1/2 the Hb is normal
which is sufficient to inhibit polymerization.
-Sickle cell Hb confers resistance to malaria.The
parasite spends part of its reproductive cycle in
the erythrocyte. Possibly, the increased fragility
of the cells disrupts the life cycle.
- heterozygotes are evidently more fit in regions
where malaria exists.
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Other types of genetic defects:
Thalassemias-one or more chains are not
produced
- One of the genes is deleted or has
undergone a nonsense mutation
- All genes may be present but
transcription may be blocked
Effects of thalassemias:
- -thalassemia. Individuals
homozygous for this defect must use fetal
() chains to make 2 2 Hb. These
individuals usually don’t survive to
maturity. There are milder cases where
transcription in only partially inhibited.
The text describes another type of
thalassemia.
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Hemoglobin Mutants: Missense, Nonsense, and Frameshift
The top line above shows the DNA code and amino acid
sequence for the first 29 amino acids of the beta globin
chain. The next four lines show the same information for
four, different mutants. The changed base and amino
acid(s) shown in red.
HbS and HbC are both missense mutants at the #6 amino
acid position substituting valine or lysine for glutamic acid.
Hb Thalassemia is a general name for a reduction in the
amount of hemoglobin produced. The first HbThal mutant
shown has a Stop or Termination codon (TAG) at position
#17 in place of the codon for lysine. The second HbThal
mutant results from the deletion of two adjacent adenines at
position #8 causing a shift in the reading frame to produce
a series of missense amino acids terminating at a now inphase, early termination codon.
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