Myoglobin and Hemoglobin

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Myoglobin and Hemoglobin

• BL4010 10.12.06

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Myoglobin Hemoglobin

• Objectives
• Identify biological functions.
• Identify parts of Mb their roles in O2
  transport.
• Identify how Hb differs from Mb.

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Myoglobin

• O2 transport, storage in cells.
• Two parts protein and heme prosthetic group.
• Protein
• 155 amino acids, 17 kDa.
• Compact, globin fold.
• 75 helix.

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Heme

• Heme is composed of porphyrin and Fe2.
• Porphyrin is non-polar, except two proprionate
  groups.
• Porphyrin binds O2, CO.
• CO 10,000x tighter than O2.

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Heme Ligands

• Fe is 6-coordinate.
• Four N of heme group
• One N from proximal His.
• One from H2O or O2.
• Can also bind CO.

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Heme Protein Interaction

• Heme stabilizes protein fold.
• Binds through hf interactions
• Prooprionate groups on surface.

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Myoglobin

• Protein completely surrounds heme.
• Function of protein
• ? heme solubility.
• ? heme oxidation
• (Fe2 ? Fe3)
• metmyoglobin inactive.
• ? CO binding.

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O2 vs. CO binding.

• CO binds tightly linear.
• O2 binds less tightly, bent structure.
• Distal His forces bent binding of both, weakens
  CO binding.

Distal His
Proximal His
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Hemoglobin

• Tetrameric protein
• Dimer of dimers, (ab)2
• a,b chains resemble Mb.
• Each chain contains, heme, prox. histidine.
• Each binds 1 eq. O2.
• ab dimer interface different from aa, bb
  interface.
• Marked by salt bridges that stabilize the deoxy
  structure.

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Oxygen Binding Curves

• Objectives.
• Understand O2 binding curve for Mb.
• Understand O2 binding curve for Hb.
• Identify mechanism of cooperative binding.

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Quantify O2 binding.

• Measure deoxy- vs. oxy by visible absorption
  (Soret band).
• Reaction is Mb O2 MbO2
• Equil. Const. given by
• Ka MbO2/MbO2
• Plot fraction bound
• Y MbO2 / (Mb MbO2)
• Recast in terms of measureable quantities
• Y pO2 / (pO2 pO2,50)

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Direct Plots

• Plot fractional saturation versus partial
  pressure of O2.
• Most relevant part of plot in range of cell pO2
  ( 15-25 mm Hg).

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Direct Plot

• Binding characterized by pO2,50.
• Partial pressure of O2 where Mb is half
  saturated.
• If lower oxygen affinity, curve shifts right if
  higher, curve shifts left.

pO2,50 3 mm Hg.
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4 Structure of Hb alters O2 binding.

• Interactions between dimers alters oxygen
  binding.
• Direct plot shows Hb has lower affinity than Mb.
• Sets up delivery system.
• O2 bound by Hb in lungs released in tissues.

Mb
Hb
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Cooperative regulation

• Hb oxygen binding
• Start binding with given affinity in deoxy state,
  subsequent binding enhances affinity.
• Defines positive cooperative regulation.
• Only seen in multi-domain proteins.
• Hill coefficient number of interacting
  subunits. Advantage binding is more sensitive to
  small changes in ligand.

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Molecular Model

• In deoxy state, Fe out of heme plane domed.
• Bind O2, moves Fe.
• This moves proximal His and its helix.
• Moving helix alters a/b interface.
• Deoxy Tense (T)
• Oxy Relaxed (R)

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T ? R Structural Changes.

• T form
• H143 - D94 in b1.
• K40 - H146 CO2-.
• R141 - D126 a2/a1.

• R form
• All are broken.

oxy
deoxy
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Small changes translate to large movements.
Deoxy State
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Small changes translate to large movements.
Oxy state
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Modifying O2 Binding in Hb

• Objectives.
• Identify allosteric effectors
• Describe molecular basis of each.

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Bohr Effect

• Oxygen affinity sensitive to pH.
• ? pH ? pO2,50 (lowers sensitivity).
• D94 ? H146 salt bridge in T state only.
• Excess H forms salt bridge, favors deoxy state.

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CO2

• Produced during aerobic metabolism.
• Reacts with N terminal amino carbamylation
  reaction.
• Negative charge forms salt bridge with aR141,
  stabilizes deoxy state.

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CO2 is Coupled to Bohr Effect

• In Tissue
• CO2 is bound by Hb or converted to bicarbonate by
  carbonic anhydrase.
• Buffers blood pH.
• Hb binds 2H / 4 O2 released, also buffers (Bohr
  effect).
• In Lungs
• Low pCO2 reaction reverses
• CO2 and H released from Hb,
• pO2,50 decreased (increased oxygen affinity).

From Langes Biochemistry
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Chloride Anion Binding

• Also favors T state
• Forms salt bridge with R141, V1 in T state.
• Released in R state.

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2,3-BPG

• Side product of glycolysis.
• indicates active respiration, need O2.
• Binds cationic region in T-form.
• Favors deoxy, releases O2 to tissues.
• 2,3-BPG is high, responsible for observed
  pO2,50 of 27 mm Hg.
• stripped Hb has pO2, 50 8 mm Hg.

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Another look at 2,3-BPG

• BPG acts as a wedge and drives the R state to
  the T state.
• Forces release of bound O2 in active tissue.
• BPG increases at high altitude.

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NO and Hb

• NO potent vasodilator
• Produced by Nitric Oxide Synthase (NOS).
• Arginine ? Citrulline NO
• Activates soluble guanylyl cyclase, signal
  transduction cascade.
• Reacts with Hb, which inactivates the NO (cant
  react with sGC).

• Interactions with Hb
• Binds to HbO2 to form nitrates (NO3-)
• Binds to deoxy-Hb to form iron-nitrosyl
  (Fe2-NO).
• Rapid reaction in vitro, but slow in vivo due to
  nature of blood flow along endothelium and
  diffusional barriers.

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Transport of NO by Hb

• NO also binds to Cys93 on b-chains.
• Forms S-NO bond (SNO-Hb).
• Transfers NO to glutathione, which functions as a
  storage form for NO (wont react with HbO2).

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NO transport by Hb

• Transfer reaction depends on T/R state.
• R-state promotes binding to Cys93
• SNO-Hb formed in lungs.
• NO released in T-state.
• Effectively delivers NO to vasculature.
• Vasodilation then enhances O2 delivery.

• But wait a minute
• SNO-Hb 10,000 x lower than HbO2.
• NO transfer from SNO-Hb is slow.
• Amount released cant compete with NO produced by
  NOS in erythrocytes.

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Clinical relevance

• If NOS impaired, SNO-Hb is effective transport,
  O2 delivery system.
• Therapeutic value in treatment of sickle cell?
• Use in blood substitutes?