Protein Function MyoglobinHemoglobin

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Protein FunctionMyoglobin/Hemoglobin
From Protein Data Bank PDB ID 1A3N Tame, J.,
Vallone, B. Deoxy Human Hemoglobin. 1998
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Protein functions

• Diverse functions
• Structural components of cells
• Motor proteins
• Enzymes
• Antibodies
• Hormones
• Oxygen carriers (Hemoglobin/myoglobin)
• Transport of macromolecules in blood
• Functions of proteins related to structure

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Hemoglobin/myoglobin

• O2 very insoluble in water
• Non-polar due to equal sharing of electrons in
  covalent bonds
• Body has O2 carrying proteins to overcome
  limitation of solubility
• Hemoglobin (Hb) - located in RBC
• Also transports CO2 and H
• Myoglobin (Mb) - located in muscle fibres
• Facilitates movement of O2 from blood to
  mitochondria

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Heme

• Mb and Hb able to bind O2 due to presence of Heme
  group
• Heme
• Non-peptide prosthetic group
• Consists of
• Protoporphyrin ring
• Fe2 (ferrous) ion which can form bonds on either
  side of heme plane

From Elliott, WH. Elliott, DC. (1997)
Biochemistry and Molecular Biology. Oxford
Oxford University Press. p374
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Myoglobin (Mb)

• Mb is a compact globular protein
• Heme group located in crevice
• surrounded by non-polar residues, except for 2
  histidines
• Non-polar residues protect Fe2 from oxidation to
  Fe3 (Hematin) which will not bind O2

From Stryer, LS (1988) Biochemistry (3rd Ed).
New York WH Freeman Co. p145
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Myoglobin (Mb)

• Fe2 of heme group bonded to proximal histidine
  (F8)
• Oxygen binds on other side of heme plane
• Distal histidine (E7) reduces binding space
• forces O2 to bind at an angle
• makes bond weaker so O2 more easily removed when
  required

Adapted from Stryer, LS (1988) Biochemistry
(3rd Ed). New York WH Freeman Co. p146
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Hemoglobin (Hb)

• Hb has quaternary structure
• 4 polypeptide chains held together by
  non-covalent bonds
• Each chain contains heme group located in crevice
  near exterior of molecule

Adapted from Elliott, WH. Elliott, DC. (1997)
Biochemistry and Molecular Biology. Oxford
Oxford University Press. p379
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Hemoglobin (Hb)

• Conformations of Mb and Hb chains similar despite
  only 17 homology of amino acid sequence
• Similar conformation provides heme group with
  environment to reversibly bind O2 whilst
  protected from oxidation

From Stryer, LS (1988) Biochemistry (3rd Ed).
New York WH Freeman Co. p152
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Hemoglobin (Hb)

• Despite similar conformations, quaternary
  structure of Hb confers additional biological
  functions

Myoglobin Carries O2 Binding of O2
non-cooperative Affinity for O2 independent of
pH, CO2 and 2-3 BPG
Hemoglobin Carries O2, CO2 and H Binding of O2
cooperative Affinity for O2 dependent on pH, CO2
and 2-3 BPG
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Oxygen dissociation curve

• Fractional occupation of oxygen binding sites
  (SO2)
• Oxygen dissociation curve describes relationship
  between pO2 and SO2
• Mb has higher affinity for O2 than Hb
• P50 - partial pressure at 50 saturation
• P50 Mb 1 Torr (mmHg)
• P50 Hb 26 Torr (mmHg)
• Higher P50 for Hb means PO2 need only decrease to
  26 mmHg for Hb to give up half of O2 carried
• PO2 must decrease to 1 mmHg for Mb to give up
  half of O2 carried

From Stryer, LS (1988) Biochemistry (3rd Ed).
New York WH Freeman Co. p154
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Cooperative binding of O2

• Sigmoidal character of Hb oxygen dissociation
  curve due to cooperative binding of O2
• Binding of O2 at one heme site facilitates
  binding at other heme sites
• Unloading of O2 from one heme site facilitates
  unloading from other heme sites

From Stryer, LS (1988) Biochemistry (3rd Ed).
New York WH Freeman Co. p154
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Mechanism of cooperative binding of O2

• Oxyhemoglobin - R (relaxed) form
• Deoxyhemoglobin - T (tense or taut) form
• 4 chains anchored firmly together by non-covalent
  electrostatic attractions (salt links)
• Fe2 situated outside heme plane - too big to fit

From Stryer, LS (1988) Biochemistry (3rd Ed).
New York WH Freeman Co. p160
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Mechanism of cooperative binding of O2

• On oxygenation
• Fe2 becomes smaller and moves into heme plane
  (becomes planar)
• Fe2 pulls proximal histidine with it
• Transmits conformational changes to interfaces
  between 4 chains and ruptures some salt-links
• Most difficult for first O2 to bind because
  chains held in place by most salt-links, but
  progressively easier as fewer links left to hold
  chains in place
• Results in sigmoidal binding curve

From Stryer, LS (1988) Biochemistry (3rd Ed).
New York WH Freeman Co. p160
From Stryer, LS (1988) Biochemistry (3rd Ed).
New York WH Freeman Co. p154
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Allosteric regulation of Hb

• Cooperative binding is example of allosteric
  regulation
• i.e. binding of molecule at one site affects
  activity at another site
• Activity of Hb also allosterically regulated by
• CO2
• 2-3 BPG
• H (pH)

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Allosteric regulation of Hb by CO2

• pCO2 high in metabolically active tissues
• CO2 combines with unionised amino groups on
  side-chains of Hb to form carbamate

• Carbamate negatively charged and forms
  salt-bridges which stabilise Hb in T form
• Reduces affinity of Hb for O2 and facilitates
  unloading of O2
• In lungs pCO2 low
• Carbamate yields CO2 which is given off and
  exhaled
• Affinity of Hb for O2 increased and facilitates
  loading of O2

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Allosteric regulation of Hb by pH

• pH low (i.e. H high) in metabolically active
  tissues
• H combine with negatively charged sites on
  side-chains of Hb
• Results in conformational changes which stabilise
  Hb in T form
• Reduces affinity of Hb for O2 and facilitates
  unloading of O2
• Assists in unloading O2 at tissues during
  exercise when pH is low
• In lung, pCO2 low
• carbonic anhydrase converts H (with HCO3-) to
  CO2 and H2O
• CO2 is exhaled

From Stryer, LS (1988) Biochemistry (3rd Ed).
New York WH Freeman Co. p156
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Allosteric regulation of Hb by 2-3BPG

• 2-3BPG synthesised by RBC when tissue pO2 is low
• 2-3BPG binds to central cavity of Hb
• Central cavity has amino acid sidechains with 4
  positive charges projecting in
• 2-3BPG has 4 negative charges and correct size
  and configuration to fit into cavity
• Binding of 2-3BPG stabilises T form and
  facilitates unloading of O2

Adapted from Elliott, WH. Elliott, DC. (1997)
Biochemistry and Molecular Biology. Oxford
Oxford University Press. p379
From Stryer, LS (1988) Biochemistry (3rd Ed).
New York WH Freeman Co. p157
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Protein denaturation

• Denaturation changes the physical and chemical
  properties of a protein
• Denaturation results from the disruption of the
  bonds holding the protein structure together
• Once bonds disrupted the protein can uncoil and
  form bonds other than those which provided its
  original configuration
• Protein cannot then regain original configuration