Transcript Oxygen Binding Proteins

Protein Function:
Oxygen Binding Proteins
CH 339K
Myoglobin
• Sperm Whale Myoglobin was the first protein to have its 3dimensional structure determined
– John Kendrew(1958)
– Shared the 1962 Nobel in chemistry
• Solving the structure wasn’t hard, but getting the samples was a
real achievement…
Kendrew, JC; Bodo, G; Dintzis, HM; Parrish, RG; Wyckoff, H; Phillips, DC (1958). "A threedimensional model of the myoglobin molecule obtained by x-ray analysis". Nature 181
(4610): 662–666.
Myoglobin
• Myoglobin - 17,000 daltons
(monomeric) 153 amino acids
• 8 a-helices, designated A - H
• Conjugated protein - A
conjugated protein has a nonprotein part in addition to a
polypeptide component.
Myoglobin – naming of helices
Heme
Heme Function
• The heme group is responsible for the O2 binding capacity of hemoglobin.
• The heme group consists of the planar
aromatic protoporphyrin made up of four
pyrrole rings linked by methane bridges.
• A Fe atom in its ferrous state (Fe+2) is at the
center of protoporphyrin.
Heme Binding
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Fe+2 has 6 coordination bonds, four bonded to the 4 pyrrole N atoms.
The nucleophilic N prevent oxidation of Fe+2.
The two additional binding sites are one on either side of the heme
plane.
One of these is occupied by the imidazole group of His F8. (H63 in
(SWM)
The second site can be reversibly occupied by O2, which is hydrogenbonded to another His. (His E7, H94 in SWM)
Oxygenation state can be measured
spectrophotometrically
Reflectance spectra of myoglobin (1), metmyoglobin (2) and
oxymyoglobin (3).
Absorbance Curves for Mb
CO Poisoning
Myoglobin’s affinity for carbon monoxide is ~ 60x its affinity for O2.
Hemoglobin’s affinity for carbon monoxide is ~ 230x its affinity for O2.
Autopsy photo showing characteristic skin discoloration
O2 Binding Kinetics
Reaction: Mb + O2 ⇌ MbO2
(1)
[Mb  O 2 ]
Keq 
[Mb][O2 ]
(2
[Mb  O 2 ]  Keq[Mb][O 2 ]
(3)
bound
  % bound 
(0    1)
total
(4)
(5)
[Mb  O 2 ]

[Mb]  [Mb  O 2 ]
Keq[Mb][O 2 ]

[Mb]  Keq[Mb][O 2 ]
(5)
(6)
(7)
(8)
Keq[Mb][O 2 ]

[Mb]  Keq[Mb][O 2 ]

[O2 ]
1
 [O2 ]
Keq
0.5 
[O2 ]50
1
 [O2 ]50
Keq
[O2 ]

[O2 ]50  [O2 ]
Dr. Ready finally gets
to the point!
Remember Dalton’s Law – the concentration of
a gas in a liquid …
… is proportional to the partial pressure of
that gas over the liquid
So:
[O2 ]

[O2 ]50  [O2 ]
Converts to:
pO2

p50  pO2
Hyperbolic Binding Kinetics
1.000
0.900
0.800
0.700

0.600
0.500
0.400
0.300
0.200
0.100
0.000
0
20
40
60
pO2 (m m Hg)
80
100
p50 Defines the Curve
1
0.9
0.8
0.7

0.6
0.5
2 mm
5 mm
0.4
10 mm
0.3
0.2
0.1
0
0
20
40
60
pO2
80
100
Oxygen Transport
3o structure overlap:
myoglobin, a-globin and b-globin
a-Globin (blue)
b-Globin (violet)
Myoglobin (green)
Mb vs. Hb
Hemoglobin O2 carrying capability
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Erythrocytes/ml blood: 5 billion
( 5 x 109 )
Hemoglobin/red cell: 280 million ( 2.8 x 108 )
O2 molecules/hemoglobin: 4
O2 ml blood: (5 x 109)(2.8 x 108)(4) = (5.6 x 1018)
or (5.6 x 1020) molecules of O2/100 ml blood
or ~ 0.3 g/l
or ~ 9 mM
By comparison:
• Solubility of O2 in saline: ~ 0.007 g/l
• or ~ 0.2 mM
O2 Binding – Hb vs. Mb
O2 transport capability, a comparison
Cooperativity
Substrate affinity changes with substrate
concentration
or (rephrased)
Substrate affinity changes with substrate
binding
Characteristic of (many) proteins with
multiple binding sites.
Cooperative Binding Kinetics
Reaction: Hb + nO2 ⇌ HbnO2
(1)
[Hb  nO2 ]
Keq 
[Hb][O2 ]n
[O 2 ] n
(2)

(3)
pOn2

p50n  pOn2
1
 [O 2 ] n
Keq
(5)
pO2

p50  pO2
(6)
1  
p50  pO2
pO2

p50  pO2 p50  pO2
(7)
1  
p50
p50  pO2
(8)
pO2
p50  pO2


p50
1 
p50  pO2
(9)
pO2


1   p50
Hill Equation
(10)

log
 log(pO 2 )  log(p50)
1 
(11)

log
 n * log(pO 2 )  n * log(p50) Hemoglobin
1 
Myoglobin
Cooperativity Models: Concerted
Monod, Wyman, and Changeaux (MWC) (1965)
Only T and R conformations exist
The two states are in equilibrium
T R transition involves shift in equilibrium constant
Cooperativity Models: Sequential
Koshland, Nemethy, and Filmer (KNF) (1966)
There are intermediate conformations between T and R
Intermediate conformations have intermediate binding
affinities
Change involves gradual conformational shift from more Tlike states to more R-like states
Hemoglobin T and R States
Hb is more MWC-like than KNF-like
T (Low Affinity)
R (High Affinity)
Shift from T to R – another view
Structural Basis
• O2 Bound conformation
does not permit several
intersubunit bonds
Histidine “Ratchet” locks T and R states
• Histidine at H97 of b1 fits into socket between T41 and
P44 in a2 in the T state
• In the R state, the valine side chain locks between
T38 and T41.
In the b chains, the C teminal His
makes a salt link with Asp FG1
This holds the F helix in a position
that keeps the Fe+2 out of the plain of
the heme ring
That in turn lowers the O2 affinity
Shift to the R state by the adjacent a
chain breaks salt link to C-terminal
His, which moves it out of position to
bind Asp FG1
Relaxation of F helix allows heme
Fe+2 to assume high-affinity position
Bohr Effect
• The O2 affinity of hemoglobin decreases with
decreasing pH
• Improves delivery of oxygen to the tissues
Bohr Effect
• C-terminal Histidine of the b subunits is
protonated at low pH
• His b146 can then form a salt link with
Aspb94 in the deoxy (T) conformation
• This stabilizes the T state of the protein.
Carbamate Formation
Covalent binding at the N-terminus of each subunit
• CO2 transport is improved since some CO2 is now being
carried back to the lungs directly by hemoglobin
• The release of H+ decreases pH and increases the Bohr
effect
• Negatively charged carbamylated N-termini form salt link
to the positive charge on Arginine a141. This salt link
stabilizes the deoxy (T) form of the molecule and favors O2
release.
2,3-Bisphosphoglycerate Binding
Combined
Effects
CO2 , BPG and pH
are all allosteric
effectors of
hemoglobin.
Fetal Hb
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Fetal hemoglobin has 2 α and 2 g chains
The g chain is 72% identical to the b chain.
A His involved in binding to 2,3-BPG is replaced with
Ser. Thus, fetal Hb has two less + charges than adult
Hb.
The binding affinity of fetal hemoglobin for 2,3-BPG is
significantly lower than that of adult hemoglobin
Thus, the O2 saturation capacity of fetal hemoglobin
is greater than that of adult hemoglobin
This allows for the transfer of maternal O2 to the
developing fetus
Fetal Hb Binding Curve is Always to the Left
of the Maternal Hb Binding Curve
Disease From a Hemoglobin Mutation
Sickle Cell
• Misshapen cells cause
vascular occlusion
• Chronic anemia
• Periodic episodes of pain
• Autosplenectomy after
infarct
• Complications
• Infection
• Stroke
• Renal Failure
• Retinopathy
•Life expectancy much
improved since 60’s, but
still shortened: 42 ♂ 48 ♀
Sickle Cell Complications
Above: dactylitis
Below: swollen, scarred
spleen
Sickle Cell
• Cause: Glu b6 changed to Valine by gene mutation
• Hydrophobic residue binds to pocket on adjacent b
chain of deoxygenated form
• ~5% of American blacks carry gene
• This is not a neutral mutation
Geographic Distribution of HbS
Malaria Belt
Heterozygote Advantage
• Heterozygous individuals in Nigeria had a
29% higher likelihood of surviving to
adulthood than homozygous normals.
• The gene is maintained in the population by
selection against both homozygotes.
Other O2 Binding Proteins (w/o Heme)
Other O2 Binding Proteins
• Hemocyanins
– Molluscs and some arthropods
– Copper acts as binding metal
– Cu(I) (colorless)  Cu(II) (blue)
– 75 kDal monomers (arthropods)
• Each monomer has 2 Cu, binds 1 O2
– Form dimers or hexamers
– Polymers form very large complexes
Hemocyanin structures
A. 24mer from Eurypelma (a tarantula) B. Single subunit from Limulus
(horseshoe crab) C. 20 x 8mer from Haliotis (Abalone) (each individual
polypeptide is an 8-fold repeat) d. C-terminal subunit from Octopus.
Other O2 Binding Proteins
• Hemerythrins
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Sipunculids, brachiopods, priapulids, bacteria
Binuclear iron center
Fe(II)  Fe(III)
13-14 kDal monomers
• Each monomer has 2 Fe, binds 1 O2
– Form (most often) octamers
– Not cooperative
Fe
\
O-H
+ O=O


/
Fe
A (deoxy)
Fe-O-O
\
:
O··H
/
Fe
B


Fe-O-OH
\
O
/
Fe
C (oxy)
Sipunculid
Priapulid
Brachiopod
Hemerythrin
O2 Binding Sites
Another Heme Protein
That Doesn’t Bind O2
The Disease - Chagas
Symptoms
• Acute Phase (weeks
to months)
• Chronic Phase (10-20
years post-infection)
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Swelling at the infection site
Fever
Fatigue
Rash
Body aches
Headache
Loss of appetite
Nausea, diarrhea or vomiting
Swollen glands
Enlargement of your liver or
spleen
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Irregular heartbeat
Inflamed, enlarged heart
(cardiomyopathy)
Congestive heart failure
Sudden cardiac arrest
Difficulty swallowing due to
enlarged esophagus
Abdominal pain or constipation
due to enlarged colon
Chagas Complications
Acute Stage –
swelling at bite
location
Chronic Stage megacolon
Chronic Stage –
cardiomyopathy,
congestive heart failure
The Agent: Trypanosoma cruzi
The Vector – Cone Nosed Bugs
Rhodnius prolixus (Tropical)
Triatoma gerstaeckeri (Local)
The Protein - Nitrophorin
• dimer of 20 kdal monomers
• Heme contains Fe+3
• salivary glands of Triatomid bugs
• bind nitrous oxide (NO)
Nitrophorin Action –
slightly dramatized
• Ravenous insect climbs onto face of
peacefully sleeping human victim
• Inserts hideous proboscis into helpless
victim’s flesh
• Nitrophorin, with NO bound, is injected
into the bite wound
• In the alkaline environment of the ghastly wound, NO is
released
• NO acts as vasodilator, prevents platelet accumulation
• Empty binding site on nitrophorin binds histamine
• Antihistamine effect prevents irritation to wake hapless
blood donor.
Lest you think this is all theoretical…
Potential future distribution in
Texas
From Emerging Infectious Diseases (2003) 9(1):
103-105