Effect of Protein Structure on Oxygen Binding

1
Effect of Protein Structure on Oxygen Binding

• The binding of oxygen to the heme iron is greatly
  influenced by the structure of the protein.
• Notice how there is a cavity in which oxygen
  binds, which is created by valine (located on
  helix E) and phenylalanine (located in the loop
  between helices C and D).
• In free solution, carbon monoxide will bind to
  heme, more than 20,000 times better than oxygen.
  However, when the heme is bound to myoglobin, it
  only binds 200 times better.
• Because of the electronic configuration of
  oxygen, it binds to the heme in a bent
  conformation, and a histidine residue (located on
  helix E) makes a favorable hydrogen bond with it.
• Because of the electronic configuration of carbon
  monoxide, it can bind only in a linear
  conformation. Therefore, the histidine
  sterically hinders its binding to the heme,
  reducing its affinity.
• There is no clear pathway for oxygen binding to
  the heme. Molecular motions (breathing) of the
  protein create transient routes to allow oxygen
  to bind or leave. The rotation of the distal
  histidine is one important molecular motion.

Distal histidine
Proximal histidine
2
Histidine F8 is termed the proximal ligand to the
heme. There is another very important histidine
amino acid in the oxygen binding pocket. His E7
helps to form the binding pocket. Heme will bind
CO 20,000 times more tightly than oxygen, but in
myoglobin, it is only about 200 times. Because
of steric repulsion with HisE7, the binding of CO
is not optimal. It is bent rather than straight
on, which is unfavorable for the electronic
structure of CO. The normal, favorable binding
of oxygen to heme is at an angle. 60 or 120
from the plane of the heme.
3
Oxygen binding alters myoglobin conformation.
Normally, the iron is not exactly in the plane of
the porphyrin ring. It is displaced 0.055 nm
towards the His proximal ligand. Binding of
oxygen pulls the heme closer into the porphyrin
ring. In turn the histidine is pulled along,
distorting the shape of the a-helix. In
hemoglobin this similar effect on oxygen binding
has profound effects on the ability of other
subunits to bind oxygen. In general, myoglobin
has an intrinsic greater affinity for oxygen than
hemoglobin at all oxygen pressures.
Deoxymyoglobin
4
Protein Ligand Associations

• The functions of myoglobin and hemoglobin are to
  bind oxygen when its concentration or partial
  pressure is relatively high, and then release it
  where it is needed, meaning where its
  concentration or partial pressure is relatively
  low.
• Figure a below represents a hypothetical binding
  curve for some ligand L to some protein. The
  greek letter q, represents the fraction or
  percent of the protein that has the ligand L
  bound to it.
• The curve that is denoted is hyperbolic, and is
  described by the equation y x / (x z). Y
  represents the fraction of ligand bound, x
  represents the ligand concentration, and z
  represents the dissociation constant (Kd) or the
  inverse of the association constant (Ka).
• Notice that when x is very large with respect to
  z, the equation approaches the limit y 1. This
  is the asymptote of the curve. Notice that when x
  is significantly smaller than z, that the
  equation represents a line with slope (1/z).
• Extrapolation to the x axis from the point on the
  curve that represents 50 occupancy gives the
  dissociation constant.

5
Partial Pressure

• The adjacent figure shows that actual curve for
  oxygen binding to myoglobin.
• Notice the nomenclature. The oxygen
  concentration (which is the ligand) is referred
  to as pO2, which means partial pressure.
  Normally, partial pressure is measured in units
  of kilopascals (kPa). Sometimes it is seen to be
  measured in mm Hg or torr (1 mm Hg 1 torr).
• The concentration of a gas or volatile substance
  in solution is proportional to its partial
  pressure in the gas phase above the solution.
• Oxygen accounts for about 21 of the total
  atmospheric mixture of gases. At sea level, the
  total pressure is 1 atmosphere, or 760 mm Hg, or
  100 kPa.
• At standard pressure, myoglobin is fully
  saturated with oxygen.

6
Oxygen Binding to Hemoglobin

• Oxygen binding to hemoglobin is distinctly
  different from its binding to myoglobin. Notice
  that the curve is not hyperbolic it is
  sigmoidal. This is a sign of cooperativity.
• The job of hemoglobin is to bind oxygen
  efficiently in the lungs, where the partial
  pressure of oxyen is about 13.3 kPa however, it
  needs to release it in the tissues, where the
  partial pressure is about 5 kPa.
• Notice how myoglobin is almost fully saturated at
  5 kPa pO2. Therefore, myoglobin would not
  release its bound oxygen to tissues.
• From looking at the sigmoidal curve for
  hemoglobin, it can be seen that it binds oxygen
  relatively weakly at low oxygen concentrations,
  but strongly at high oxygen concentrations.
• This behavior is referred to as allostery.
  Hemoglobin is an allosteric protein.

7
Allostery

• An allosteric protein is one in which the binding
  of a ligand to one site affects the binding
  properties of a ligand to another site on the
  same protein.
• If the same ligand modulates further binding of
  itself to the protein, this is called homotropic
  allostery. If a ligand modulates binding of a
  molecule that is different from itself, this is
  called heterotropic allostery.
• Allostery is frequently observed in multimeric
  proteins, wherein the binding of one ligand to
  one of the subunits, affects the binding of
  ligands to the other subunits.
• Allostery can be positive or it can be negative.
• The sigmoidal curve that is seen can be
  envisioned to be a hybrid curve of two normal
  hyperbolic curves, wherein the dissociation
  constants are different. The high affinity site
  would have a low dissociaiton constant, while the
  low affinity site would have a high dissociation
  constant.

8
Models for Allostery

• Two models for the cooperative binding of ligands
  to proteins with multiple binding sites have been
  advanced.
• The MWC (Monod, Wyman, and Changeux) modelwhich
  is designated the concerted modelassumes that
  each subunit is identical and can exist in two
  different conformations or states. The two
  states have different affinities for the ligand
  however, all subunits within one protein can
  exist in only one of the two states. The binding
  of ligand to a subunit in the low affinity state,
  results in a conformational change that places it
  in the high affinity state. All other subunits,
  even though they do not have a bound ligand, must
  follow suit.
• In the Koshland model, which is the sequential
  model, ligand binding can induce a conformational
  change in just one subunit. This will then make
  a similar change in an adjacent subunit, making
  the binding of a second ligand more likely.

concerted
sequential
9
T and R State of Hemoglobin

• Below are the two major conformations of
  hemoglobin as predicted by the models for
  allosteric activation.
• Oxygen will bind to hemoglobin in either state
  however, it has a signficantly higher affinity
  for hemoglobin in the R state.
• In the absence of oxygen, hemoglobin is more
  stable in the T state, and is therefore the
  predominant form of deoxyhemoglobin. R stands
  for relaxed, while T stands for tense, since this
  is stabilized by a greater number of ion pairs.
• Upon a conformational change from the T state to
  the R state, ion pairs are broken mainly between
  the a1b2 subunits.

10
Subunit Contacts in Hemoglobin

• The strongest interactions in the hemoglobin
  tetramer are between the two subunits that make
  up the protomer unit (a1b1 or a2b2). These
  interactions are shaded in blue in the lower left
  figure.
• The yellow shaded area represents sliding
  contacts. These are contacts made between two
  unlike subunits (a1b2 or a2b1), which undergo
  significant changes upon going from one state to
  the other.
• The a1b1 protomer rotates on top of the a2b2
  protomer about an imaginary pivot in the center
  of the protein, breaking some of the ion pairs in
  the yellow shaded region.

11
The Hill Relationship

• The Hill equation log(q/(1-q)) n L logKd
• Notice that the Hill equation is an equation of a
  line (y mx b), wherein n (the number of
  subunits or binding sites) is the slope.
• The experimentally determined slope does not
  reflect the number of binding sites however. It
  reflects, instead, the degree of cooperativity.
  Because of this, n is usually labeled nH, which
  is called the Hill coefficient.
• For hemoglobin, the hill coefficient is 3,
  although there are four subunits that can bind
  oxygen.
• When there is no cooperativity at all, nH 1.
• The theoretical upper limit for cooperativity
  would be when nH n.
• To adapt the Hill equation to oxygen binding to
  hemoglobin, pO2 should replace L, and P50
  should replace Kd.

12
Heme Structural Changes

• In the T state, the porphyrin is slightly
  puckered. The histidine is not optimally ligated
  to the iron. Because of steric problems, it is
  about 8 from being perpendicular.
• As in myoglobin, the iron lies out of the plane
  of the porphyrin, but moves towards the plane on
  oxygen binding.
• The subsequent distortion of the helix to which
  His F8 is bound, necessitates that the subunits
  adjust their conformations.
• These changes lead to adjusments in the ion pairs
  at the a1b2 interface.