In the 0.95 Å cholesterol oxidase structure, a narrow tunnel
extends from the exterior surface of the molecule to the buried
active site cavity. The tunnel is situated between secondary structure
elements of the FAD-binding domain and the substrate-binding domain.
The tunnel only becomes evident at 0.95 Å resolution due
primarily to the ability to visualize many more multiple conformations
in the higher resolution structure. The tunnel in this structure
is narrow in width, essentially only wide enough to accommodate
a single water molecule. The side chain conformations are correlated,
that is when one changes conformation, the rest must follow for
steric reasons. Occupancy of one conformation forms an open tunnel,
occupancy of the second conformation closes the tunnel. The opening
and closing appears to be gated by the side chain of Asn485, which
stabilizes the reduced cofactor through an N-H···p
interaction as described above. We proposed that upon reduction
of the cofactor, the asparagine side chain moves closer to stabilize
the extra electron density on the FAD; this movement forces a
series of side chain movements in the region of the tunnel, thereby
forming the tunnel and providing access to the active site for
molecular oxygen (Figure 4a and b).
Figure 4: Surface representation
of cholesterol oxidase (pdbentry 1MXT) showing the proposed oxygen
tunnels in the open and closed positions. The O2 entry point and
the steroid binding cavity (SBC) are labeled. The yellow arrows
identify the gating residue whose conformation restricts access
from the oxygen tunnel to the steroid binding cavity. The (a)
open and (b) closed conformations.
The tunnel is wide enough to house only a single string of water
molecules linked to each other by a hydrogen bond network. Furthermore,
the residues lining the tunnel are all hydrophobic in nature;
this results in a very unreactive environment. The size of the
tunnel, the nature of the residues that line it, the gating aspects
of the tunnel and the location of its endpoint at the pyrimidine
ring of the FAD cofactor suggest that it may function as an entry
point for molecular oxygen during the oxidative half–reaction
of the enzymes. The timing of oxygen access may be required to
prevent radical peroxidation of the cholest-5-en-3-one in the
active site. The kinetics are consistent with the formation of
a ternary complex of oxygen, steroid, and enzyme. Moreover, the
entrance to the tunnel is on a solvent-accessible face of the
enzyme when it is bound to the lipid bilayer. A second reason
that a separate binding pathway may be required for oxygen is
that the steroid is delivered to the enzyme from the lipid bilayer
interface, and oxygen is delivered from the aqueous interface.
