Chapter 7 - Protein Function -- PowerPoint

I. Myoglobin and Hemoglobin Function

A. Structure

  • 1. Mb consists of a single polypeptide containing 153 aa residues; secondary structure is all a-helix
    • 8 a-helices labeled A-H
    • 44Å x 44Å x 25Å
    • Heme bound to hydrophobic cleft

    2. Hb is a tetrameric protein of two different types of subunits, a2,b2, which are very similar to myoglobin, Mb.

    •

    Hb carries O2 through blood system: O2 solubility in blood plasma is 10-4 M but with Hb solubility is

    0.01 M, about the same as air. Mb serves to increase O2 solubility in cells/tissues

    A. Heme: protoporphyrin IX (a tetrapyrolle) with centrally bound Fe+2 ion (Fe doesn't change oxidation state during Hb function) Fig. 7-2

    B. Mb Function: Oxygen binding and transport in tissues

    1. O2 binding to Mb is described by a simple equilibrium
  • Mb + O2 = Mb-O2 ==> Keq = ([Mb] [O2]) / [Mb-O2]

    O2 dissociation from Mb commonly described by its fractional saturation, YO2, and [O2] in partial pressure, pO2

    and substitution from the equilibrium expression gives

    The O2 binding curve for Mb is simple hyperbolic function described by the equation above
    p50 is the pO2 at which Mb is 50% saturated, and from the equation above p50 = K

    •

    C. Hb Function: Oxygen binding and transport via blood from lungs to tissue

    1. Hb + 4 O2 = Hb(O2)4 or in more general terms Hb + nO2= Hb(O2)n
  • in the latter case the O2 binding equation becomes:
    •

    which is also known as the Hill Equation

    2. Hb binding curve for O2 as a function of pO2 is not hyperbolic but sigmoidal ==> binding of O2 by Hb changes significantly over physiological range of pO2 values. Fig. 7-7
    • Mb binds O2 under conditions in which Hb releases it
    • the two form an O2 transport system from lungs to cells.

    3. A Sigmoidal binding curve is an indication of cooperative interactions among Hb subunits ==> once one subunit binds O2 the other 3 also bind O2.

    4. Derivation of the Hill Equation assumes complete cooperativity ==> all subunits bind ligand (O2 for Hb) at the same time. This is never completely true, but the closer n comes to the number of subunits the more cooperative the binding is. In the case of Hb, n = 2.8-3.0, close to the maximum value of 4, and O2 binding to Hb is very cooperative.

    5. Measuring Hill Coefficient, n:

  • the Hill Equation can can be rearranged to:

    and so plotting log{YO2 / (1 - YO2)} vs. logpO2 gives a line with slope = n

    Fig. 7-8: Hill Plot for Mb (straight line with n = 1

    6. Hb has a complex Hill Plot:

    • n = 1 at very low pO2 when most Hb molecules don't have any O2 bound and at high pO2 when each Hb has at least 3 O2 bound.
    • At intermediate pO2's, n = 2.8 - 3 =~ high cooperativity
    • Extrapolation of n=1 portions of the plot show 100 fold greater O2 affinity (p50 = 0.3) for last O2 bound compared to first (pO2 = 30)

    The general form of the Hill Equation for an enzyme catalyzed reaction is:

    •

    D. Carbon Dioxide Transport and the Bohr Effect Fig. 7-12

    1. When Hb binds O2 it releases protons; this is called the Bohr Effect
  • Hb (O2)n Hx + O2 == Hb(O2)n+ x[H+]

    2. This means that lower pH induces Hb to release O2

    3. CO2 produced in cells by respiration is converted to carbonic acid by carbonic anhydrase in red blood cells, and carbonic acid dissociates to produce bicarbonate ion and H+; thus the CO2 stimulates the relase of O2 from Hb.

    4. CO2 also decreases Hb O2 affinity by binding prefferentially to the deoxy form which has lower O2 affinity than the oxy. Fig. 7-13

    •

    E. Effect of BPG Bisphosphoglycerate (diphosphoglycerate or DPG) Fig. 7-14

    1. Binds to Hb in blood reduding its O2 affinity

    2. DPG binds to deoxy Hb but only weakly to oxy Hb ==> it tabilizes the deoxy conformation reducing the overall affinity of Hb for O2

    3. DPG thus shifts the O2 binding curve so Hb will release its O2 in capillaries. This is in addition to the effect of CO2.

    4. Adaptation to high altitude

    • over weeks adaptation results in increased Hb and red blood cells
    • overnight adaptation results in increased [DPG] which decreases O2 affinity of Hb allowing it to release more O2 at the capillaries.

    F. Hb is an Allosteric Protein whose affinity for O2 is altered by a conformational change described by one of two models

    1. Symmetry Model (Monod, Wyman, Changeux) Fig. 7-19
    • all subunits within a molecule are in either the T-state with low affinity for O2 or in the R-State with high affinity
    • O2 binding promotes the conversion from T-State to R-State

    2. Sequential Model (D. Koshland) Fig. 7-20

    • conformational changes occur sequentially as more O2 bindings sites are occupied
    • sequential model allows for negative cooperativity

    G. Hb has a T-state to R-state transition -- Fig. 7-5,6,9,10,11

    1. Deoxy-Hb is in the T-state

    2. Oxy-Hb is in the R-State

    3. Transition from T-State to R-state

    • involves both tertiary and quaternary structural changes
    • Fe++ moves into Heme plane plulling His-Helix F
    • a1-b2 and a 2-b1 interfaces move WRT one another
    • Ionic bonds in a chains and in b chains are broken in the R-state decreasing pK's of sidechains and causing the release of H+ upon oxygenation