Protein Function/Ligand Binding Myoglobin and Hemoglobin Structure and Function

• Ligand binding is of fundamental importance in biochemical phenomena. (REVIEW hyperbolic binding equilibrium from bioenergetics lecture notes.)
  
  
• Heme (Fe protoporphyrin IX) in myoglobin and hemoglobin binds O₂ reversibly, without oxidation of the Fe⁺² which is required for O₂ binding.
  
  
• Myoglobin and hemoglobin's structures and ligand binding properties have evolved differently for the different functions of the two proteins, and the structure-function relationships are very well understood.
  • Mb is monomeric, 1 O₂ binding site per molecule, hyperbolic binding curve (no cooperativity)
  • Hb is tetrameric, 4 O₂ binding sites per molecule, sigmoid binding curve
    • indicative of cooperative ligand binding (structural communication between different binding sites by conformational changes)
    • Hb is thus an allosteric protein
      • R state ("oxy" conformation, high O₂ binding affinity) stabilized by O₂ binding (O₂ is a homotropic effector)
      • T state ("deoxy" conformation, low O₂ binding affinity) stabilized by binding of protons, CO₂, and/or 2,3-bisphosphoglycerate (2,3-BPG) (all heterotropic effectors)
    • allosteric regulation of O₂ binding to Hb is important to enhance the ability of Hb to RELEASE O₂ in the tissues.
      
      
      
• Hill Plot [plot of log {q / (1 - q)} vs. log [L}] permits measurement of Hill coefficient (n_H), a way to assess degree of cooperativity in ligand binding.
  
  
• Hb also transports some CO₂ from tissues to lungs in the form of carbamate adducts at amino termini of chains.
  
  
• 2.3-BPG is needed in human erythrocytes (red blood cells) to reduce O₂ binding affinity enough to get effective release of O₂ in tissues.
  • 2,3-BPG binds in central cavity of Hb (stoichiometry 1 BPG/Hb tetramer)
    
    
• Fetal Hb (HbF) has different 4^o structure from adult HbA (a₂g₂ vs. a₂b₂)
  • Sequence difference between g and b reduces HbF's affinity for 2,3-BPG, thus increasing its affinity for O₂ under physiological conditions.
    
    
• Two major models for cooperative ligand binding (allosteric proteins)
  • MWC (concerted, symmetry) model -- NO "hybrid" 4^o structures with some subunits in R state and some in T state
  • KNF (sequential) model -- does allow for "hybrid" 4^o structures with some subunits in R state and some in T state
    
    
• Examples of mutant hemoglobins are described whose properties are altered in one of the following ways:
  • mutation in heme binding pocket leads to loss of heme
  • mutation disrupts tertiary structure of a subunits
  • mutation stabilizes methemoglobin (Fe⁺³ oxidation state of heme in Hb)
  • mutation stabilizes the R state, or stabilizes the T state, compared to their stabilities in normal HbA

LIGAND BINDING:
The essence of protein function/action is BINDING (recognition of and interaction with other molecules).

• Proteins bind (just a few examples):
  • small molecules & ions (including protons!)
  • other proteins
  • nucleic acids (DNA, RNA)
  • membranes
  • polysaccharides
    
    
• BINDING results from specific, usually NONCOVALENT interactions between complementary molecular surfaces
  • SHAPE complementarity (lots of van der Waals interactions, atoms pack against each other)
  • CHEMICAL complementarity (hydrogen bonds, salt linkages, hydrophobic interactions)

LIGAND: a molecule or ion (usually small) that's bound by another molecule (usually large, e.g. a protein)

Binding equilibrium (a review from Bioenergetics lectures):

• concept fundamental in biochemistry
  • same general concept as proton binding/dissociation
  • Protons are ligands! K_d for proton dissociation is acid dissociation constant.
• Analysis of binding equilibria requires a few simple algebraic equations:
• chemical equation for the dissociation of the ligand (L) from the protein (P)
   
• expression for the equilibrium dissociation constant K_d for the reaction
  (Concentrations of free protein [P], free ligand [L], and P•L complex [PL] in this expression are the equilibrium concentrations.)
  

• Useful parameter for plotting data:  q (greek letter theta). q is fraction of total binding sites on the protein ([P]_total) that are actually OCCUPIED by ligand under the given conditions, [occupied sites]/[total sites]:
  

• range of values for q
  • q varies from 0 (all sites empty, at [L] = 0) to 1.0 (max. q is with all sites occupied, "saturation" conditions, [L] >> K_d)
  • q = 1.0 isn't experimentally quite achievable, since approach to site saturation is asymptotic.

Combining expressions for Kd and q gives . Equation of a rectangular hyperbola.  Concentration of ligand, [L], for which q = 0.5 is Kd. Kd = [L] that gives 50% of the binding sites occupied (thus 1 - q  =  0.5 = fraction of sites that are empty) difficult to accurately estimate q = 0.5 from inspection of graph of raw signal vs. [L], because curve approaches saturation asymptotically, but if q is plotted (which means max. signal was KNOWN), max. occurs at q = 1.0, so obviously q = 0.5 is easy to locate on graph to determine Kd.

• A linear transformation of the binding equation can be used to determine K_d by a graphical extrapolation to infinite [L] (, or a curve-fitting program on a computer or calculator can fit the data to the equation for a hyperbola and give a value for K_d that best fits the data.
• Example of a linear transformation of the binding equation:

  or

• Such equations give linear plots, which makes determination of K_d more accurate.

• Remember that K_association = 1/K_dissociation
• NOTE: K_d is the concentration of ligand needed to HALF-SATURATE the binding sites.

MYOGLOBIN AND HEMOGLOBIN:
PARADIGMS OF LIGAND BINDING AND PROTEIN FUNCTION AND REGULATION

• Oxygen
  • required for oxidative metabolism and energy production in most cells
  • O₂ the terminal electron acceptor in aerobic metabolism
    • used to oxidize dietary substrates to carbon dioxide, yielding energy
• Hemoglobin (Hb) used by vertebrates
  • to transport O₂ to tissues from lungs (or gills) and
  • to transport CO₂ from tissues to lungs (or gills)
• Myoglobin (Mb) used in some tissues, notably muscle,
  • as a storage reserve of O₂ and
  • for intracellular transport of O₂
• Mb monomeric protein (153 amino acid residues)
• Hb is a tetramer of 2 a chains (141 residues each) and 2 b chains (146 residues each). 

Tertiary Structure of Myoglobin

• Myoglobin ("Mb"):
  • Structure
    • monomeric (1 polypeptide chain) intracellular protein (mainly in muscle cells), 153 amino acid residues, M_r 16,700
    • first 3-dimensional structure of a protein (Kendrew et al., 1950's)
    • Mb structure: 78% a helical (the other 22% in b turns and short loops, no b sheet at all)
    • GLOBIN tertiary structure ("globin fold"):
      • 8 a helices designated by letters A-H, in an N to C terminal direction, and connections between helices are referred to as "AB", "CD", etc.
      • Residues in each helix are numbered within that helix, to permit consistent references to the same conserved residues or structural regions in different globin proteins with slightly different lengths of polypeptide chains.
        • For example, the "proximal His" residue, involved in binding of the heme in all the globins, is residue #93 in Mb, residue #87 in Hba, and residue #92 in Hbb. However, because it is the 8th residue in the F helix in all 3 polypeptide chains, we can refer to it as "HisF8" and immediately know which residue we mean in all 3 chains.
    • Mb structure illustrates many principles of folding and stability of water-soluble globular proteins:
      • all peptide bonds trans and planar
      • provided first direct experimental evidence for existence of a helices in proteins
      • 4 Pro residues, 3 in bends (4th in an a helix); other bends with Ser, Thr, and Asn residues, sizes compatible with tight turns
      • interior atoms densely packed
      • lots of hydrophobic residues, with most hydrophobic R groups buried
      • all but 2 polar R groups at outer surface, all hydrated
        
        
• Function
  • function of Mb is to bind O₂ that has been delivered to tissues by the hemoglobin in the blood,
    • 1) storing the O₂ until it's needed as terminal electron acceptor for energy metabolism, and
    • 2) transporting it within the cell ("molecular bucket brigade", O₂ dissociates from one Mb molecule and binds to the next)
  • Mb and Hb both bind O₂ with the help of a heme prosthetic group**
    **prosthetic group: a metal ion or an organic or metalloorganic compound other than an amino acid that's tightly bound to a protein (sometimes covalently, sometimes TIGHT but noncovalent binding) and is required for the protein's function/activity
  • TERMINOLOGY: Apoprotein = protein without its prosthetic group; holoprotein = protein WITH the bound prosthetic group (holoprotein = "whole", if that helps!)
    apoprotein + prosthetic group <==> holoprotein
    
    
• HEME: complex organometallic compound, iron-protoporphyrin IX, with Fe⁺² bound
  
  
• Fig. 6-17 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000)  Heme structure, Fe-protoporphyrin IX
  • Fe ion has 6 coordination bonds, 4 in plane of porphyrin to N atoms, 2 perpendicular to it
  • 1 perpendicular bond from Fe goes to the eN of the imidazole side chain of "proximal His" residue in protein (His F8, residue #93 in Mb sequence)
  • other perpendicular bond goes to the ligand (O₂) when O₂ is bound
    • O=O forms a coordination bond from 1 O atom to the heme Fe²⁺, and a hydrogen bond from the other O atom to the eN of the distal His residue.

• Here's another figure showing heme structure, without (left) and with (right) bound O₂:
  Note the 2 propionic acid side chains (terminal carboxyl groups with red O atoms).
• The IRON (blue in figure below) is in the ferrous state (Fe⁺²).  OXYGEN (red in figure below) binds reversibly to the Fe⁺² , but the iron is NOT oxidized to Fe⁺³. 
  • Mb or Hb in which the iron has been oxidized chemically to Fe⁺³ does not bind oxygen; instead, the Fe⁺³ forms of Mb and Hb ("metmyoglobin" and "methemoglobin") bind H₂O in the sixth coordination position of the iron (where the O₂ would bind to the heme Fe⁺²), but are INACTIVE for the normal function of Mb and Hb.

Deoxy Heme Chime structure deoxyheme Note in Chime structure that the iron atom is slightly OUT OF THE PLANE of the heme.

Oxy Heme Chime structure oxyheme Note in Chime structure that the O2 has "pulled" the iron atom INTO THE PLANE of the heme.

• Heme binds to Mb in a narrow hydrophobic crevice (you saw that in the Mb structure in online problem set).
• Where would you expect the protoporphyrin's two propionic acid (propionate-) sidechains to be located relative to the crevice/protein interior/protein surface?

• Fig. 6-16 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000) Myoglobin structure (the "globin fold")

• the heme prosthetic group is shown in red
  a) backbone only, in ribbon diagram
  b) "mesh" image emphasizing protein surface
  c) surface contour image, useful for visualizing surface pockets where ligands might bind
  d) ribbon diagram with hydrophobic side chains (Leu, Ile, Val, and Phe) shown in blue ball & stick
  e) space-filling model, again with hydrophobic side chains in blue (mostly buried)

• function of globin protein component: preventing the Fe(II) from oxidizing to Fe(III),which can't bind O₂.
  (Fe⁺² protoporphyrin IX is the form that can bind O₂; Fe⁺³ heme can bind H₂O, but not O₂; if Fe gets oxidized in Mb or Hb, the oxidized form -- which can't bind O₂ -- is called metMb or metHb.)
  • NOTE: There are many other proteins in cells with heme prosthetic groups, including an important class of proteins called cytochromes (e.g., cytochrome c).  Many of these other heme proteins function in reduction/oxidation systems (e.g. the mitochondrial and chloroplast electron transport systems), in which function of the protein requires that heme Fe DOES undergo reversible oxidation/reduction as part of its function.
  • However, Mb and Hb are NOT redox proteins -- the heme Fe must stay in the Fe⁺² state to bind O₂.
  • Protein structure in Mb and Hb crucial to prevent oxidation of the Fe by the O₂; bulky hydrophobic residues in the heme-binding pocket and presence of the DISTAL HISTIDINE (His E7) on the side of the heme where O₂ binds protect Fe⁺² from oxidation.
    
• Fig. 7-5c (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): O₂ binding to heme in myoglobin (and hemoglobin) -- key amino acid residues around the heme.
  One coordination bond from the heme Fe²⁺ goes to proximal His (His F8), which is thus directly involved in heme binding.
  Chime structure of oxyheme showing heme binding by proximal His (His F8)
  The bound O₂ (O=O) forms a coordination bond from 1 O atom to the opposite side ("distal" side) of the heme Fe²⁺, and there's a hydrogen bond from the other O atom of O₂ to the eN of the distal His residue. Distal His (HisE7) is directly involved in O₂ binding. Distal His and bulky hydrophobic R groups of Val and Phe help protect Fe²⁺ from oxidation by bound O₂.

• Chime routine for myoglobin
  

Hemoglobin

Fig. 6-23 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000) Quaternary structure of deoxyhemoglobin (hemoglobin without O₂ bound).    Chime routine: hemoglobin

• heterotetramer: 2 a subunits (gray and light blue) + 2 b subunits (pink and dark blue)
  (NOTE: designation of individual polypeptide chains with greek letters has nothing whatever to do with their secondary structural elements!)
• a and b subunits are homologous to each other and to myoglobin
  All 3 have evolutionarily related primary structures (amino acid sequences evolved/diverged from the same common ancestral protein) and the same overall folding pattern
  • primary structures of the different globin polypeptide chains have diverged over the course of evolutionary history, but
  • the crucial residues have been conserved in the amino acid sequence:
    • for heme and oxygen binding (e.g., the proximal and distal histidinyl residues) and
    • for maintenance of the overall "globin fold" (tertiary structure)
• quaternary structure (arrangement of subunits) makes it clear that the nonidentical subunits fit together as if Hb were composed of 2 heterodimers, (ab)₁ and (ab)₂ : (ab)₁ + (ab)₂ < == > (ab)₂
  • These identical structural "units" (the two ab units) are called PROTOMERS.
  • In order to distinguish between the 2 identical ab protomers in discussing the quaternary structure and conformational changes, the two protomers are often referred to as (ab)₁ and (ab)₂. The component subunits in each protomer, respectively, would be a₁ and b₁ , and a₂ and b₂ . (To avoid confusion with stoichiometric numbers, sometimes those individual subunit designations and the protomer designations are written as superscripts instead of subscripts.)
• Heme prosthetic groups (1 per subunit, so 4 per Hb tetramer) in red (ball & stick rendering) -- the 4 hemes are far apart

 

Oxygen Binding to Myoglobin

• The binding of O₂ to Mb follows a simple HYPERBOLIC saturation curve.  The fractional saturation, q, is defined by the equations below:

• Units of pO₂ on plot above are torr. Gas partial pressure units: 1 torr = 1 mm Hg = 0.133 kilopascals (kPa)
• The partial pressure of O₂, pO₂ = fraction of gas phase that is O₂, another way to express conc. of O₂.
• P₅₀ = partial pressure of O₂ required to give 50% saturation (pO₂ when half the binding sites are occupied and half are empty). 
• Note: [Mb] = [free myoglobin] = [empty binding sites]; [MbO₂] = [myoglobin with bound O₂] = [occupied sites].
• Myoglobin has just one O₂ binding site per protein molecule.

Oxygen Binding to Hemoglobin

• The O₂ saturation curve of Hb is SIGMOIDAL (S-shaped). 
  • Such a sigmoid saturation curve is diagnostic of COOPERATIVE BINDING ("COOPERATIVITY"), as a result of "communication" between different ligand binding sites on the same multimeric protein molecule. 
• Initially, Hb is in a low affinity T-STATE. 
• Binding of O₂ causes conformational change in Hb, converting it to the high affinity R-STATE.  
  • sigmoid saturation curve = a composite of
    • a low affinity curve at low O₂ concentrations and
    • a high affinity curve at high O₂ concentrations.

• Hemoglobin an example of an allosteric protein (from Greek "allos" = "other", and "stereos" = "shape").
  • binding of a ligand to one site on the (multi-subunit) protein affects the binding properties of another site on the same protein molecule (Figure on right below is Fig. 7-12 from Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000)

• The cooperative binding of O₂ to Hb is an example of an ALLOSTERIC EFFECT: the binding of one ligand to a protein influences the affinities for ligand of the other binding sites on the protein. 
• The ligands may be the same, as here (O₂ binding affects affinities of other sites on same protein molecule for O₂), or different (see below).
  • If the interacting sites all bind the same ligand (e.g., O₂ binding affecting O₂ affinity at a different site), that's a homotropic interaction, or a homotropic effect.
  • If the interacting sites bind different ligands (e.g., H⁺ binding at a proton binding site affecting O₂ affinity at the O₂ site -- see below), that's a heterotropic interaction, or a heterotropic effect.
  • The ligands are called effectors or modulators.

The Functional/Physiological Significance of the Sigmoidal Oxygen Binding Curve

• Hb has 2 major functions:
  1. transport of O₂ from lungs to tissues
  2. transport of some CO₂ (produced as a metabolic waste product in the tissues) from tissues to lungs.
• Hb transport of O₂ from lungs to tissues --
  2 components of this function:
  • binding O₂ in the lungs
  • releasing O₂ in the tissues
• O₂ binding curves below (units of pO₂ are torrs; gas partial pressure units: 1 torr = 1 mm Hg = 0.133 kPa) illustrate functional significance of sigmoid O₂ saturation curve of Hb:

While both Mb and Hb would be saturated with O2 at partial pressure of O2 in lungs, only hemoglobin can release significant amounts of O2 at partial pressure of O2 present in the tissues.  Cooperative binding enhances the ability of Hb to unload its O2 "cargo" in the tissues. In fact, the O2 released by Hb can then be bound by Mb for O2 storage in those tissues (e.g., muscle), that have significant amounts of Mb. Mb still binds O2 very tightly at the pO2 in the tissues where Hb releases most of its "payload". Typical pO2 in tissues is about ~25 torr or about 3.5 kPa

The Hill Plot

• Hill equation (describes binding of n molecules of O₂ to Hb by an equation similar to the one shown above):

  

. 

• Linear form of Hill equation (NOTE error in this equation in earlier printings of Lehninger Principles of Biochemistry; corrections posted on textbook website):
  • can be used to assess cooperativity in binding in the form of the Hill Coefficient, n_H
    
    

• Hill Plot: a plot of log {q / (1 - q)} vs. log [L]. (Fig. 7-13, Nelson & Cox Principles of Biochemistry, 3rd ed., 2000):
  • Mb: Hill plot yields a straight line with n  = 1.0
    • slope of 1 indicates that there's no communication between binding sites (no cooperativity).
    • Of course, Mb only has 1 O₂ binding site per molecule (no inter-site communication possible)
  • For Hb, Hill plot is not a straight line, but composed of three parts:
    • At low and high pO₂ the plot has a slope of 1.
      • These parts of the plot represent conditions where the first (T state) or last (R state) O₂ molecules are being added/bound.
      • Under these conditions the Hb behaves as through it had a single binding affinity (low affinity when it's all in T state, high affinity when it's all in R state), with no communication between sites on the different subunits.

At intermediate values of  pO2  there is a slope > 1. This represents the "switch" region of O2 concentration, in which as a result of cooperative ligand binding, the Hb molecules are undergoing the conformational change from the low affinity to the high affinity conformation. The value of the slope at the point on the plot where pO2 = P50 (where q = 1-q = 0.5, so [occupied sites] = [empty sites], and log {q /(1-q)} = 0) gives nH, the Hill coefficient. For Hb, nH is about 2.8-3.5, depending on conditions (pH, etc.)

• Meaning of the Hill Coefficient:
  • n_H = 1: there is no observable communication between sites, no cooperativity.
  • n_H < 1 ("negative cooperativity"): binding of ligand to one site decreases affinity of other site(s) for ligand. Well-documented cases of negative cooperativity are rare but do exist. A more common explanation for values of n_H < 1 is various kinds of artifacts, e.g. protein preparation is not homogeneous.
  • n_H > 1 ("positive cooperativity", or just "cooperative binding"): binding of ligand to one site increases affinity of other site(s) for ligand.
  • The maximal possible value for n_H for hemoglobin would be 4, the number of subunits (actually the number of binding sites for that ligand), but Hill coefficient is always less than this maximal value. If n_H = number of subunits, that's the maximum possible positive cooperativity, meaning no partially saturated molecules exist -- either all sites on a molecule are empty (T state) or all sites on a molecule are occupied (R state).

   

Hemoglobin Structure and Cooperative O₂ Binding

• Fig. 7-8 (Nelson & Cox, Lehninger Principles of Biochemistry, 2000): Contacts between the subunits within protomers where the strongest subunit interactions occur, in both deoxy- and oxyhemoglobin (T state and R state).

The differences in O₂ affinity between T-State (deoxy) and R-State (oxy) Hb can be understood in terms of the changes in quaternary structure that accompany the conversion of deoxy Hb to oxy Hb. 
• Major changes occur not between a and b subunits of same protomer, but at the interfaces (contacts) between the a₁b₁ protomer and the a₂b₂ protomer -- the contacts between a₁ and b₂ (and, symmetrically, between a₂ and b₁)

• Fig. 7-10 (Nelson & Cox, Lehninger Principles of Biochemistry, 2000): Structural differences between T state and R state, highlighting loss of several salt links between protomers in going from T state to R state; the two protomers rotate about 15o relative to each other, as well as "sliding" slightly in going from T to R.
  NOTE difference in size of central cavity (relevant to 2,3-BPG binding; see below).

• Fig. 7-11 (Nelson & Cox, Lehninger Principles of Biochemistry, 2000): Change in tertiary structure of a Hb subunit on O₂ binding triggered by shift in position of the F helix when O₂ binds and "pulls" Fe into the plane of the heme, thus pulling the proximal His (His 8) and thus moving the whole F helix.

• Overall changes can be appreciated from this animation
(oxy5). You can distinguish the oxy conformation because of the red O₂ bound to the blue iron.


• Shift from deoxy to oxy conformation arises from the fact that
  • in deoxy Hb the Fe lies out of plane of heme ring, but
  • when O₂ binding occurs, the Fe moves into plane of heme ring.
(oxy1).


• Because proximal His (His F8) is bound to the Fe, it moves also, making F helix move.
(oxy2).


• Movement of F helix alters tertiary structure of that individual subunit.
  (oxy3)
  
  
• Tertiary structural change has an effect on interactions between subunits at interfaces.
(oxy4).


• Changes in interactions at the protomer interfaces ultimately lead to shift of entire tetramer from deoxy to oxy conformation, a quaternary structural change.
(oxy5).

 

• Once the other subunits have shifted to the oxy conformation (quaternary structural change), the affinity of the unoccupied sites for O₂ increases because there are no steric blocks to O₂ binding, i.e., the conformational changes have already taken place.
   
• Because of the way the Hb subunits are packed together, change in tertiary structure of just one subunit in tetramer from the deoxy to oxy conformation does not trigger the quaternary change -- the quaternary switch requires that O₂ be bound to at least one subunit on each protomer, as indicated in this schematic diagram (figure adapted from Ackers et al., 1992, Science 255, 54-63)(PDF of same figure).
  • In fact, if O₂ binding to just one subunit on a tetramer were sufficient to trigger the quaternary switch, converting whole tetramer to the high affinity (R) state, the Hill coefficient of hemoglobin would be 4, the maximum possible value.
  • Such an "all-or-none" switch would represent a pure "concerted" or "symmetry" model of allosteric interactions (also called the MWC model, from the scientists who originally proposed it, Monod, Wyman and Changeux -- see below) , but the MWC model is too simple to describe real situation in hemoglobin.

The Bohr Effect: effect of binding of protons (H⁺) and CO₂ on O₂ binding affinity of Hb
first discovered by Danish physiologist Christian Bohr (1855-1911)

• Protons are negative heterotropic effectors (allosteric inhibitors) of O₂ binding to Hb.

• The origin of the Bohr effect lies in the fact that deoxy Hb has several functional groups that are weaker acids than they are in oxy Hb, so O₂ binding reduces H⁺ affinity, and H⁺ binding reduces O₂ affinity:
  
• A major contribution to the Bohr effect involves C-terminal His residue of each b subunit. 
• In the deoxy state, this His forms a salt bridge to Asp 94, IF the His ring is protonated.  
• Salt bridge stabilizes protonated form of the His, causing a higher pK_a (weaker acid) in deoxy state (T state).
• Salt bridge does not form in the oxy state (R state).  Thus protonation of His-146 favors the deoxy conformation (bohr: animation of salt bridge formation upon deoxygenation).

Carbon Dioxide Transport

• CO₂ is a negative heterotropic effector (allosteric inhibitor) of O₂ binding to Hb.
• presence of CO₂ in the tissues reduces affinity of Hb for O₂ (favors deoxy, T state) in two ways:

1. CO₂ lowers the pH (Bohr effect)
2. CO₂ participates in formation of carbamates by the N-terminal a-amino groups of Hb:
   
   
   
   • Formation of carbamate releases H⁺, which contributes to the Bohr effect, and
   • the negative charge introduced on the protein (carbamate) allows formation of additional salt bridges, but only in the deoxy state (T state).  Thus, carbamate formation (CO₂ binding) favors the deoxy state.

2,3-Bisphosphoglycerate:  another negative heteroptropic effector of O₂ binding to Hb

• [By what kind of linkages/bonds are the two phosphate groups connected to the glycerate backbone? What functional groups can undergo condensation reactions to produce the bis-phosphorylated product, 2,3-BPG?]

• Human red blood cell contains high concentrations of 2,3-bisphosphoglycerate (2,3-BPG), sometimes called "2,3-diphosphoglycerate" (DPG).
• 2,3-BPG binds strongly to the deoxy form of Hb (T state), but only very weakly to oxy form (R state).
• 2,3-BPG binds to ONE SITE, in the CENTRAL CAVITY, on the Hb tetramer (so what is the stoichimetry of 2,3-BPG binding to the Hb tetramer?)
  • Central cavity only large enough to accommodate 2,3-BPG in the T form (deoxy)
  • Smaller central cavity in the more compact R form (oxy) is too small, so
  • 2,3-BPG favors/stabilizes the T form, reducing the O₂ binding affinity of Hb (shifts binding curve to the right).
• Look at the structure of 2,3-BPG. Which type of noncovalent bonds would you predict would be involved in its binding to a protein?

• Salt links (ionic interactions) between 2,3-BPG's negatively charged groups (2 phosphates and a carboxyl group) and positively charged groups on the protein.
  • several basic groups (Lys and His R groups, and the N-terminal a-amino group) on BOTH b chains
  • Salt links effectively cross-link the quaternary structure across the central cavity in the T state.

Hb(BPG) [T state] + O₂ <==> Hb(O₂) [R state] + BPG

• Thus, 2,3-BPG binding favors the deoxy (T) conformation.
  

• Fig. 10.23 from Berg, Tymoczko & Stryer, Biochemistry, 5th ed., 2001): Mode of binding of 2,3-BPG to central cavity of human deoxyhemoglobin (T state) (bpg: Chime structure of hemoglobin in presence and absence of 2,3-BPG).

• In fact, if human hemoglobin did NOT have its O₂ affinity reduced by bound BPG, we could not effectively release O₂ to our tissues. (See 0 mM BPG binding curve on figure below, or in Nelson & Cox Fig. 7-16.)
• Fig. 7-25 from L. Stryer, Biochemistry, 4th ed., 1995:

• NOTE: 2,3-BPG is derived metabolically from 1,3-BPG, an important metabolic intermediate in the glycolytic pathway. The allosteric effector of Hb's O₂-binding affinity is the 2,3-BPG, not 1,3-BPG.
• ALSO NOTE: Not all animals use 2,3-BPG to regulate the O₂ binding affinity of their hemoglobins. In some mammals (e.g., cattle, sheep, and cats) the affinity is lower to begin with and 2,3-BPG has little effect. Birds use a different organophosphate molecule, inositol pentaphosphate (IPP) or inositol hexaphosphate (IHP), as a regulatory molecule to reduce the O₂ affinity of their hemoglobin; fish and most amphibians use ATP for this regulatory function. The organic phosphate heterotropic effectors bind in the central cavity of these other species' hemoglobins, also.

The importance of 2,3-bisphosphoglycerate (2,3-BPG) as an allosteric regulator of hemoglobin's O₂ affinity
Figure below is similar to Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000, Fig. 7-16. Arterial pO₂ = pO₂ in lungs; venous pO₂ = pO₂ in tissues

• High altitude adaptation
  • Human adaptation to high altitude is a complex physiological process that involves many events, including
    • an increase in number of erythrocytes and also
    • an increase in the amount of hemoglobin per erythrocyte, which may take several weeks to accomplish.
  • One event that occurs within 24 hours is an increase in the content of 2,3-BPG in the erythrocyte.

Fetal O₂ transport
Arterial pO₂ = pO₂ in lungs; venous pO₂ = pO₂ in tissues (placenta in this case)

• Fetus obtains O₂ by diffusion across placenta.
  • This diffusion is aided by the fact that fetal Hb (HbF) has a higher affinity for O₂ than does maternal (adult) HbA under physiological conditions (in red blood cells, with 2,3-BPG is present in both maternal and fetal cells).
  • The affinity difference is NOT due to inherently tighter O₂ binding by fetal hemoglobin subunits, but rather due to a difference in the fractional saturation of the hemoglobin with 2,3-BPG for fetal vs. maternal hemoglobin.

• How would having less BPG bound (lower affinity for BPG) affect the O₂ binding affinity of HbF?
  
  Would you expect the O₂ binding affinities of HbF and HbA to differ from each other in the absence of any 2,3-BPG? (Would maternal Hb be able to effectively transfer its O₂ to HbF if human erthyrocytes lacked 2,3-BPG?)

• In practical terms,
  • at comparable 2,3-BPG concentrations in maternal and fetal erythrocytes, HbF binds O₂ more tightly than HbA, so
  • O₂ can be released from maternal Hb and bound by fetal Hb in the placenta.
• Thus, maternal HbA can "deliver" O₂ to the HbF through the placenta.

The Physiological Transport of O₂ and CO₂

• How do all these allosteric effects combine to allow Hb to carry O₂ from the lungs (gills) to the tissues and to carry CO₂ from the tissues to the lungs (gills)?
  • As shown below, Hb stripped of all its allosteric effectors has too high an affinity for O₂ to allow effective transport of O₂ to tissues.  
  • However, the presence of both CO₂ in the tissues and 2,3-BPG in the red blood cells creates a situation in which O₂ is efficiently transported from lung to tissue.

We can summarize these events as follows:

1. In lungs, partial pressure of O₂ is high, which overcomes any negative allosteric effects and causes complete oxygenation of Hb in arterial blood (oxyHb conformation predominates).
2. As erythrocytes with HbO₂ enter tissue capillaries, higher CO₂ and proton concentrations (lower pH) combine to favor deoxy conformation (T state) and release of O₂.
3. The presence of 2,3-BPG aids the delivery of O₂ in the tissues by favoring the deoxy conformation.
4. Deoxy Hb binds CO₂ in the tissues.
5. The deoxy Hb (carrying CO₂) returns to lungs, where pH is higher, the O₂ concentration is higher and the CO₂ concentration is lower.  All these factors favor reversal of carbamate formation (loss of bound CO₂, which gets exhaled), deprotonation of His 146 and the binding of O₂ again (R state, oxyHb).

Allosteric regulation/terminology -- review

• Cooperative interactions (allosteric interactions) [Greek: "allos"="other", "stereos" = space] occur when binding of one ligand at a specific site is influenced by binding of another ligand, which is called an "allosteric effector" or "modulator".  The second site is also called an allosteric site.
• If the ligands are identical (e.g., binding of O₂ at one site on Hb influences the binding affinity for O₂ of another site), the effect is called a homotropic effect.
  • If the homotropic effector increases the binding affinity for the same kind of ligand at other sites, the effect is referred to as a positive homotropic effect (e.g., O₂ in the hemoglobin system increases the O₂ binding affinity of other sites).
• If the ligands are different (e.g., binding of protons at one site on Hb influencing the binding affinity for O₂ at a different site on the protein), the effect is called a heterotropic effect.
  • If the heterotropic effector decreases the binding affinity for the primary ligand, it is referred to as a negative heterotropic effector, or an allosteric inhibitor (e.g., protons, or CO₂, or 2,3-BPG; all are negative heterotropic effectors of O₂ binding to hemoglobin).
  • If the heterotropic effector increases the binding affinity for the primary ligand, it is referred to as a positive heterotropic effector, or an allosteric activator (no examples in the hemoglobin system, but plenty of examples in enzyme regulation).

Models for Allostery, Cooperative Ligand Binding (equilibria between high-affinity and low-affinity conformations)

• Fig. 7-14 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000): 2 models for allosteric proteins
  CIRCLES = low affinity, or inactive (T state) conformations; SQUARES = high affinity, or active (R state) tertiary conformations of individual subunits; subunits with bound ligand are shaded.
  
  • MWC (Monod-Wyman-Changeux model) = "symmetry" or "concerted" model
    • if ligand binds to one subunit, all subunits undergo the R <--> T transition simultaneously
    • there are no "hybrid" protein molecules with some subunits in R state and some in T state.
  • KNF (Koshland-Nemethy-Filmer model) = "sequential" model
    • ligand binding can induce a change in conformation of an individual subunit in a multimeric protein without all the rest of the subunits switching conformations
    • "hybrid" R/T multimers can exist
• MWC can be regarded as just the two "all-or-none" extremes of the KNF model, without any of the intermediate (hybrid) quaternary states
• Different proteins are best described by different models, with Hb cooperative behavior being best modeled by a blend of the two models.

Mutant Hemoglobins

• Several hundred mutant hemoglobins are known to exist. 
• In most, a single amino acid replacement occurs in either the a or b chain of normal HbA. 
• Many of these changes cause no known effect, but several lead to pathologies associated with abnormal O₂ transport.
• Example: HbS, sickle cell hemoglobin  (hemoglobinS)
  • single amino acid substitution of a Val instead of the normal Glu at position 6 of the b chain.
  • This seemingly innocuous change places a hydrophobic sidechain on surface of the protein, TWO per Hb tetramer. 
  • In deoxy conformation the Val sidechain of a b chain in one HbS binds to hydrophobic pocket on surface of b chain of another HbS tetramer. This leads to
    • polymer formation (fibers) / precipitation of the deoxy HbS, and thus
    • red cell lysis and anemia
  • Hydrophobic pocket is lacking in oxy conformation of HbS, so no problem with oxyHbS, and much less problem with heterozygous individuals than with individuals homozygous for the HbS mutation.
  • Individuals with the HbS mutation have a small but significant selective advantage in resistance to malaria, accounting for the persistence of such a deleterious mutation in populations in parts of the world where malaria is endemic.

Mutant Hemoglobins
What can be learned from experiments of nature?

• Mutant hemoglobins provide unique opportunities to probe structure-function relations in a protein.
• There are nearly 500 known mutant hemoglobins and >95% represent single amino acid substitutions.
• About 5% of the population carries a variant hemoglobin.
• Some mutant hemoglobins cause serious illness.
• The structure of hemoglobin is so delicately balanced that small changes can render the mutant protein nonfunctional.
• The following images were generated using HbA (2hhb.pdb, C. Fronticelli, unpublished and 1 hho.pdb, B. Shaana, J. Mol. Biol. 17, 31 (1984)), and the  Swiss-PdbViewer to introduce the various mutations.  Thus, the images are not derived from actual crystal structures but represent approximations to what those structures would probably look like.

Mutations that disrupt the tertiary structure of a subunit

• In b chain two helices (red and green below) pass so close together that there is only room for the H sidechain of Gly between them.
• In Hb Savannah [Gly(24)bVal], presence of Val sidechain forces the two helices apart,
• disrupting tertiary structure of b chain, and thus
• leading to an unstable hemoglobin molecule.

This is Hb A with Gly	This is Hb Savannah with Val

Mutations that stabilize Methemoglobin

• In order for hemoglobin to reversibly transport O₂, iron must remain in ferric (Fe⁺²) state.
• Oxidizing iron to Fe⁺³ produces methemoglobin, which does not transport O₂.
• (Red blood cells contains enzymes that can re-reduce the iron in the occasional normal HbA molecule whose iron gets oxidized.)
• All known methemoglobins arise from mutations that provide a negatively charged oxygen atom as a ligand for the iron.
• The negatively charged oxygen stabilizes iron in the Fe⁺³ state.

This is Hb Milwaukee [His(87)bGlu] in which the distal His, which normally protects the iron from becoming oxidized, has been replaced by Glu, which stabilizes the Fe+3 state.

Mutations that change the stability of the R State or the T State.

• Mutations at the a₁-b₂ interface (and thus also at the a₂-b₁ interface) often interfere with quaternary structure of hemoglobin.
• Such mutations can change the relative stabilities of hemoglobin's R and T states, thereby affecting O₂ affinity of mutant hemoglobin.
• Normal HbA has P₅₀ = 26 mm Hg (= 26 torr, or about 3.5 kPa). [P₅₀ is the pO₂ at which q = 0.5, so half of Hb's O₂ binding sites are occupied.]

Crocodile Hemoglobin

• Crocodiles can remain under water for up to one hour and usually kill their prey by drowning them. 
• How do crocodile's tissues get O₂ while submerged? 
• Unlike deep diving whales, which use myoglobin to store O₂ in muscle, crocodiles rely on a unique allosteric effector of Hb to ensure delivery of O₂ to tissues. 
• While submerged, metabolism produces CO₂, which is converted to HCO₃^- and it is the HCO₃^- that acts as the allosteric effector by binding to residues at the a₁b₂ interface in deoxy Hb and stabilizing the deoxy Hb. 
• This unique allosteric effect is only found in crocodile Hb. 
• Crocodile Hb does not bind BPG due to mutations in the BPG-binding pocket