Chap. 5A Protein Function
Topics
• Reversible Binding of a Protein to a Ligand:
Oxygen-binding Proteins
• The heme group
• Myoglobin
• Hemoglobin
• Sickle-cell Anemia
Fig. 5-19. Normal (a) and sickle-cell
anemia (b) erythrocytes.
Intro. to Ligand-binding Proteins
Ligands are molecules that can be bound reversibly by a
protein. Ligands can be any type of molecule, including another
protein. Proteins that bind ligands do so at sequences called the
binding site. The binding site is complementary in shape to the
ligand that is bound. The degree of complementarity determines
the binding specificity and strength. Most proteins undergo
conformational changes on binding to a ligand. Changes can be
small (i.e., “breathing”), or can be major. In a multisubunit
protein, a conformational change in one subunit can affect the
conformation of other subunits. Enzymes also bind to
complementary molecules. However, in addition to binding,
enzymes chemically transform these components. The binding
site on an enzyme is referred to as the active, or catalytic
site. The molecule acted on by the enzyme is referred to as
the substrate.
Evolution of the Heme Group
Molecular oxygen (O2) is only slightly soluble in aqueous solution.
It cannot be carried to tissues in sufficient quantity if it is simply
dissolved in blood plasma. Diffusion of O2 through the tissues is
also ineffective over distances of greater than a few millimeters.
For these reasons the evolution of large, multicellular animals
depended on the evolution of proteins that could transport (e.g.,
hemoglobin) and store (e.g., myoglobin) oxygen. Still, something
other than a protein per se is needed for O2 transport as none of
the 20 standard amino acids can bind oxygen well. It is thought
that this need led to the evolution of a bound prosthetic group
know as heme which is functional in reversible O2 binding.
Structure and Properties of the Heme Group
Heme consists of a complex organic ring structure known as
protoporphyrin IX to which is bound a single iron atom in its
ferrous (Fe2+) state (Fig. 5-1). The iron atom has six coordination
bonds, four to nitrogen atoms that are part of the flat porphyrin
ring (Fig. 5-1 d) and two that are perpendicular to the porphyrin
(Fig. 5-1 b, c, & d). The coordinated nitrogen atoms help prevent
conversion of the heme iron to the ferric (Fe3+) state which does
not bind O2. Heme is found in many oxygen-transporting proteins
as well as in proteins such as the cytochromes, which participate
in electron-transfer reactions
Binding of the Heme Group to Myoglobin
The heme group of myoglobin (and hemoglobin) is sequestered within
a crevice, or pocket, in the protein (Refer to Fig. 4-16). The
burying of the heme prevents a reaction that would occur with free
heme in solution in which one O2 binds to two sandwiched heme
groups and oxidizes iron to Fe3+. In myoglobin (and hemoglobin),
only one of the perpendicular coordination positions of heme Fe2+
actually is unoccupied and available for O2 binding (Fig. 5-2). The
other position is occupied by a His residue of the polypeptide chain
that is commonly called the proximal histidine.
When O2 binds to heme the electronic
properties of the iron atom change and
solutions containing the heme turn from a
dark purple to a bright red color. Note that
other small molecules such as carbon
monoxide (CO), nitric oxide (NO), and
cyanide ion (CN-) can replace O2 in the open
coordination position. As discussed below,
structural features of the heme binding
pocket interfere with the binding of these
other ligands and reduce their strength of
interaction with heme iron.
The Globin Family
The globins are a widespread family of proteins that are
commonly found in eukaryotes of all classes and even in some
bacteria. Most globins function in oxygen transport or storage,
although some are involved in the sensing of gases such as O2,
NO, and CO. At least four types of globins occur in humans and
other mammals. These include the monomeric myoglobin, which
facilitates oxygen diffusion in muscle tissue, and the tetrameric
hemoglobin, which transports oxygen in the blood. A third
monomeric globin, neuroglobin, is expressed in neurons and helps
protect the brain from hypoxia (low oxygen) and ischemia
(restricted blood supply). A fourth monomeric globin, cytoglobin,
is found in number of tissues, but its function is unknown.
The Structure of Myoglobin
Myoglobin (Mb) is a monomeric,
globular protein (153 aas, Mr 16,700)
that contains one heme prosthetic
group. There are eight -helical
segments in Mb and other globins, and
these are labeled A through H
consecutively in the sequence (Fig. 53). The bends in the molecule are
designated AB, CD, EF, and so forth,
based on the -helical segments they
connect. Globins have an unusually high
percentage of -helical structure (78%
in Mb). In our discussion of Mb (and
hemoglobin) structure and function, we
will refer to several residues in the
sequence. Note that these residues
can be numbered based on their
positions in the amino acid sequence,
or by their locations in the sequence of
a particular -helical segment. For
example, the His residue that is
coordinated to the heme group of Mb
can be referred to as His93 or His F8.
Mathematical Description of Protein-ligand
Binding (I)
The reversible binding of a protein (P) to a ligand (L) can be
described by the equilibrium expression
P + L  PL
The reaction is characterized by an equilibrium constant, Ka,
Ka = [PL]/[P][L] = ka/kd
where ka and kd are rate constants for association and
dissociation, respectively. Ka is the association constant that
describes the equilibrium between the complex and the unbound
components of the complex. The Ka provides a measure of the
affinity of the ligand for the protein. Ka has units of M-1, and the
higher the value of the Ka, the higher the affinity of the ligand
for the protein.
A rearrangement of the equilibrium equation shows that the ratio
of bound to free protein is directly proportional to the
concentration of the free ligand.
Ka[L] = [PL]/[P]
Mathematical Description of Protein-ligand
Binding (II)
The binding equilibrium can be described from the standpoint of
the fraction (theta, ) of ligand binding sites in the protein
that are occupied by ligand
 = binding sites occupied/total binding sites = [PL]/([PL] + [P])
Substituting Ka[L][P] for [PL] and rearranging terms gives
 = Ka[L][P]/(Ka[L][P] + [P]) = Ka[L]/(Ka[L] + 1) = [L]/([L] + 1/Ka)
The value of Ka can be determined graphically from a plot of 
vs the concentration of the free ligand, [L] (Fig. 5-4a, next
slide). Note that an equation of the form x = y/(y + z)
describes a rectangular hyperbola, and  therefore is a
hyperbolic function of [L]. The fraction of ligand-binding sites
occupied approaches saturation asymptotically as [L] increases.
The [L] at which half of the available ligand-binding sites are
occupied ( = 0.5) corresponds to 1/Ka.
Mathematical Description of Protein-ligand
Binding (III)
In the curve, the [L] at which half of the available ligand binding
sites are occupied is equivalent to 1/Ka, or Kd (see next slide).
The curve has a horizontal asymptote at  = 1.0 and a vertical
asymptote (not shown) at [L] = -1/Ka.
Mathematical Description of Protein-ligand
Binding (IV)
It is more common (and intuitively simpler), to consider the
dissociation constant, Kd, which is the reciprocal of Ka (Kd = 1/Ka)
and is given in units of molar concentration (M). Kd is the
equilibrium constant for the release of ligand from the complex.
Expressions analogous to those derived above can be written using
the Kd, namely
Kd = [P][L]/[PL] = kd/ka
and
= [L]/([L] + Kd)
The latter equation plots the curve shown in Fig. 5-4a in the
preceding slide.
When [L] equals Kd, half of the ligand-binding sites are occupied.
As [L] falls below Kd, progressively less of the protein has ligand
bound to it. In order for 90% of the available ligand-binding
sites to be occupied, [L] must be 9 times greater than the Kd.
Finally, note that a lower value of the Kd corresponds to a higher
affinity of the ligand for the protein (see Table 5-1, next slide).
Worked Example 5-1. Receptor-ligand
Dissociation Constants
Analysis of O2 Binding to Mb (I)
A series of equations describing the binding of O2 to Mb can be
derived from the equations used to describe the general binding
of a ligand to a protein. First, the concentration of dissolved
oxygen can be substituted for [L] into the equation relating  to
[L] and Kd, namely
 = [O2]/([O2] + Kd)
As for any ligand, Kd equals the [O2] at which half of the
available ligand-binding sites are occupied, or [O2]0.5.
 = [O2]/([O2] + [O2]0.5)
In experiments using oxygen as a ligand, it is the partial
pressure of oxygen (pO2) in the gas phase above the solution
that is varied. pO2 is easier to measure than the concentration
of O2 dissolved in solution. The concentration of a volatile
substance in solution is always proportional to the partial
pressure of the gas. So if we define the partial pressure of
oxygen at [O2]0.5 as P50, substitution into the above equation
gives
 = pO2/(pO2 + P50)
Analysis of O2 Binding to Mb (II)
A binding curve for Mb that relates  to pO2 is shown in Fig. 54b. The partial pressure of O2 in the air above the Mb solution is
expressed in kilopascals (kPa). Oxygen binds tightly to Mb, with a
P50 of only 0.26 kPa.
Protein Structure Affects Ligand Binding (I)
The specificity with which heme binds its various ligands is altered
when heme is a component of Mb (and hemoglobin). For example,
CO binds to free heme molecules more than 20,000 times better
than O2 (that is the Kd or P50 for CO binding to free heme is
more than 20,000 times lower than that for O2). However, CO
only binds about 200 times better than O2 when the heme is
bound to Mb. This difference may be partly explained by steric
hindrance (next slide). When O2 binds to free heme, the axis of
the oxygen molecule is positioned at an angle to the Fe-O bond
(Fig. 5-5 a). In contrast, when CO binds to free heme, the Fe,
C, and O atoms lie in a straight line (Fig. 5-5 b). In both cases,
the binding alignment reflects the geometry of the hybrid orbitals
in each ligand. (Continued on the next slide).
Protein Structure Affects Ligand Binding (II)
In Mb, His64 (His E7, the distal histidine)
on the O2-binding side of the heme is too
far away to coordinate with the heme
iron, but it does interact with ligands
bound to heme (Fig. 5-5 c). In fact, His
E7 may help preclude the linear binding of
CO, providing one explanation for the
selectively diminished binding of CO to
heme in Mb (and hemoglobin). The binding
of O2 to heme in Mb depends on molecular
motions, “breathing,” in the structure of
the polypeptide chain. The heme group is
buried so deeply in folded Mb that there
is no direct path for O2 to the
coordination site. In order for O2 to
reach its binding site in heme, side-chains
of Mb must move out of the way and
produce transient cavities in the structure
through which O2 can move.