Oxygen Binding Proteins

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					Protein Function:
Oxygen Binding Proteins

CH 339K
    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 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 three-
  dimensional 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 non-
  protein 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
•   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 hydrogen-
    bonded 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
            [Mb  O 2 ]
(1)   Keq 
            [Mb][O 2 ]

(2    [Mb  O 2 ]  Keq[Mb][ O 2 ]
                    bound
(3)     % bound           (0    1)
                     total
             [Mb  O 2 ]
      
(4)       [Mb]  [Mb  O 2 ]

            Keq[Mb ][ O 2 ]
(5)   
         [Mb]  Keq[ Mb][ O 2 ]
            Keq[Mb ][ O 2 ]
(5)   
         [Mb]  Keq[ Mb][ O 2 ]
              [O 2 ]
(6)   
            1
                [O 2 ]
           Keq
(7)              [O 2 ]50
      0.5 
               1
                   [O 2 ]50
              Keq                 Dr. Ready finally gets
                                  to the point!
(8)             [O 2 ]
       
          [O 2 ]50  [O 2 ]
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:
               [O 2 ]
      
         [O 2 ]50  [O 2 ]

Converts to:

            pO 2
      
         p50  pO 2
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    80   100
                         pO2 (m m Hg)
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   80   100
                            pO2
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
•   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
                 [Hb  nO 2 ]
    (1)    Keq 
                 [Hb][O 2 ] n

                  [O2 ]n
    (2)    
                 1
                     [O2 ]n
                Keq

                 pO n
    (3)           2

              p50n  pO n
                        2
                   pO 2
         
(5)             p50  pO 2
              p50  pO 2    pO 2
(6)    1              
              p50  pO 2 p50  pO 2
                   p50
(7)    1  
                p50  pO 2
                pO 2                  Hill Equation
             p50  pO 2
(8)         
       1       p50
              p50  pO 2
             pO 2
(9)         
       1   p50
             
(10)    log       log(pO2 )  log(p50)         Myoglobin
            1 
             
(11)    log       n * log(pO2 )  n * log(p50) Hemoglobin
            1 
  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 T-
like 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
•   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
  –   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
            \      + O=O      \ :          \
              O-H             O··H           O
            /               /             /
           Fe               Fe             Fe
           A (deoxy)        B              C (oxy)
Sipunculid   Priapulid   Brachiopod
Hemerythrin
O2 Binding Sites
Another Heme Protein
That Doesn’t Bind O2
The Disease - Chagas
    Symptoms
• Acute Phase (weeks                 • Chronic Phase (10-20
  to months)                           years post-infection)
•   Swelling at the infection site   •   Irregular heartbeat
•   Fever                            •   Inflamed, enlarged heart
•   Fatigue                              (cardiomyopathy)
•   Rash                             •   Congestive heart failure
•   Body aches                       •   Sudden cardiac arrest
•   Headache                         •   Difficulty swallowing due to
                                         enlarged esophagus
•   Loss of appetite
                                     •   Abdominal pain or constipation
•   Nausea, diarrhea or vomiting
                                         due to enlarged colon
•   Swollen glands
•   Enlargement of your liver or
    spleen
Chagas Complications

                Acute Stage –
               swelling at bite
                      location




      Chronic Stage -             Chronic Stage –
      megacolon                   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

				
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posted:11/8/2012
language:English
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