Protein Function: Oxygen Binding Proteins

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Protein FunctionOxygen Binding Proteins

• CH 339K

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Myoglobin

• Sperm Whale Myoglobin was the first protein to
  have its 3-dimensional structure determined
• John Kendrew(1958)
• Shared the 1962 Nobel in chemistry
• Solving the structure wasnt hard, but getting
  the samples was a real achievement

Kendrew, JC Bodo, G Dintzis, HM Parrish, RG
Wyckoff, H Phillips, DC (1958). "A
three-dimensional model of the myoglobin molecule
obtained by x-ray analysis". Nature 181 (4610)
662666.
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Myoglobin

• Myoglobin - 17,000 daltons (monomeric) 153 amino
  acids
• 8 a-helices, designated A - H
• Conjugated protein - A conjugated protein has a
  non-protein part in addition to a polypeptide
  component.

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Myoglobin naming of helices
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Heme
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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 (Fe2) is at the
  center of protoporphyrin.

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Heme Binding

• Fe2 has 6 coordination bonds, four bonded to the
  4 pyrrole N atoms. The nucleophilic N prevent
  oxidation of Fe2.
• 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 hydrogen-bonded to another His. (His E7,
  H94 in SWM)

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Oxygenation state can be measured
spectrophotometrically
Reflectance spectra of myoglobin (1), 
metmyoglobin (2) and oxymyoglobin (3).
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Absorbance Curves for Mb
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CO Poisoning
Myoglobins affinity for carbon monoxide is 60x
its affinity for O2. Hemoglobins affinity for
carbon monoxide is 230x its affinity for O2.
Autopsy photo showing characteristic skin
discoloration
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O2 Binding Kinetics
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Reaction Mb O2 ? Mb?O2
(1) (2 (3) (4) (5)
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(5) (6) (7) (8)
Dr. Ready finally gets to the point!
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Remember Daltons Law the concentration of a
gas in a liquid
is proportional to the partial pressure of
that gas over the liquid
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So
Converts to
Big blue arrows are usually a clue to the student
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Hyperbolic Binding Kinetics
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p50 Defines the Curve
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Oxygen Transport
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3o structure overlap myoglobin, a-globin and
b-globin
a-Globin (blue) b-Globin (violet) Myoglobin
(green)
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Mb vs. Hb
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Hemoglobin O2 carrying capability

• 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

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O2 Binding Hb vs. Mb
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O2 transport capability, a comparison
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Cooperativity

• Substrate affinity changes with substrate
  concentration
• or (rephrased)
• Substrate affinity changes with substrate binding
• Characteristic of (many) proteins with multiple
  binding sites.

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Cooperative Binding Kinetics
Reaction Hb nO2 ? Hb?nO2
(1) (2) (3)
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(5) (6) (7) (8) (9) (10) (11)
Hill Equation
Myoglobin
Hemoglobin
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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
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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 T-like
states to more R-like states
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Hemoglobin T and R StatesHb is more MWC-like
than KNF-like
T (Low Affinity)
R (High Affinity)
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Shift from T to R another view
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• Structural Basis
• O2 Bound conformation does not permit several
  intersubunit bonds

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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.

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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 Fe2 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 Fe2 to
assume high-affinity position
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Bohr Effect

• The O2 affinity of hemoglobin decreases with
  decreasing pH
• Improves delivery of oxygen to the tissues

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Bohr Effect

• C-terminal Histidine of the ? subunits is
  protonated at low pH
• His ?146 can then form a salt link with Asp?94 in
  the deoxy (T) conformation
• This stabilizes the T state of the protein.

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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
  ?141. This salt link stabilizes the deoxy (T)
  form of the molecule and favors O2 release.

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2,3-Bisphosphoglycerate Binding
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Combined Effects
CO2 , BPG and pH are all allosteric effectors of
hemoglobin.
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Fetal Hb

• Fetal hemoglobin has 2 a 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

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Fetal Hb Binding Curve is Always to the Left of
the Maternal Hb Binding Curve
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Disease From a Hemoglobin Mutation
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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 60s, but
  still shortened 42 ? 48 ?

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Sickle Cell Complications Above
dactylitis Below swollen, scarred spleen
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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

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Geographic Distribution of HbS
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Malaria Belt
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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.

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Other O2 Binding Proteins (w/o Heme)
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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

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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.
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Other O2 Binding Proteins

• Hemerythrins
• 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 Fe-O-O Fe-O-OH
\ OO \ \
O-H ? OH ? O /
? / ? / Fe
Fe Fe A (deoxy)
B C (oxy)
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Sipunculid
Priapulid
Brachiopod
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Hemerythrin
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O2 Binding Sites
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Another Heme Protein That Doesnt Bind O2
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The Disease - Chagas
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Symptoms

• Acute Phase (weeks to months)
• 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

• Chronic Phase (10-20 years post-infection)
• 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

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Chagas Complications
Acute Stage swelling at bite location
Chronic Stage - megacolon
Chronic Stage cardiomyopathy, congestive heart
failure
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The Agent Trypanosoma cruzi
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The Vector Cone Nosed Bugs
Rhodnius prolixus (Tropical)
Triatoma gerstaeckeri (Local)
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The Protein - Nitrophorin

• dimer of 20 kdal monomers
• Heme contains Fe3
• salivary glands of Triatomid bugs
• bind nitrous oxide (NO)

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Nitrophorin Action slightly dramatized

• Ravenous insect climbs onto face of peacefully
  sleeping human victim
• Inserts hideous proboscis into helpless victims
  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.

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Lest you think this is all theoretical
Potential future distribution in Texas
From Emerging Infectious Diseases (2003) 9(1)
103-105