Protein Structure Affects How Ligands Bind

The binding of a ligand to a protein is rarely as simple
as the above equations would suggest. The interaction
is greatly affected by protein structure and is often accompanied
by conformational changes. For example,
the specificity with which heme binds its various ligands
is altered when the heme is a component of myoglobin.
Carbon monoxide binds to free heme molecules more
than 20,000 times better than does O2 (that is, the Kd
or P50 for CO binding to free heme is more than 20,000
times lower than that for O2), but it binds only about
200 times better when the heme is bound in myoglobin.
The difference may be partly explained by steric hindrance.
When O2 binds to free heme, the axis of the oxygen
molecule is positioned at an angle to the FeOO bond
(Fig. 5–5a). In contrast, when CO binds to free heme,
the Fe, C, and O atoms lie in a straight line (Fig. 5–5b).
In both cases, the binding reflects the geometry of hybrid
orbitals in each ligand. In myoglobin, His64 (His E7),
on the O2-binding side of the heme, is too far away to
coordinate with the heme iron, but it does interact with
a ligand bound to heme. This residue, called the distal
His, does not affect the binding of O2 (Fig. 5–5c) but
may preclude the linear binding of CO, providing one
explanation for the diminished binding of CO to heme
in myoglobin (and hemoglobin). A reduction in CO binding
is physiologically important, because CO is a lowlevel
byproduct of cellular metabolism. Other factors,
not yet well-defined, also seem to modulate the interaction
of heme with CO in these proteins.
The binding of O2 to the heme in myoglobin also depends
on molecular motions, or “breathing,” in the protein
structure. The heme molecule is deeply buried in
the folded polypeptide, with no direct path for oxygen
to move from the surrounding solution to the ligandbinding
site. If the protein were rigid, O2 could not enter
or leave the heme pocket at a measurable rate. However,
rapid molecular flexing of the amino acid side
chains produces transient cavities in the protein structure,
and O2 evidently makes its way in and out by moving
through these cavities. Computer simulations of
rapid structural fluctuations in myoglobin suggest that
there are many such pathways. One major route is provided
by rotation of the side chain of the distal His
(His64), which occurs on a nanosecond (109 s) time
scale. Even subtle conformational changes can be critical
for protein activity.