When a substrate binds to a cytochrome P450 enzyme, a type I change to
the UV spectrum occurs as described in the previous subsection. This
is correlated with a change in the 
ion from a LS to a HS
state. X-ray crystallography has shown that this is accompanied by the
removal of a water molecule which formed the sixth axial ligand of the
and a movement of the ion out of the plane of the haem
[101, 123]. The change in the spin state coincides with a
change in the redox potential. For example, in 
the
redox potential changes from -300mV in the absence of a ligand to
-173mV in the presence of camphor. This makes the first reduction of
the energetically favourable and the reaction cycle proceeds as
described in Subsection 
, typically resulting in the
hydroxylation of the substrate which is usually hydrophobic in
nature.
Substrate analogues are similar in nature to substrates. However,
differences between these molecules and true substrates result in
differences in their binding to the active site. The crystal
structures of complexes of substrate analogues with
show that the -coordinated water molecule is retained
[100, 101, 102] and a high fraction of the LS character
of the remains. An example of the active site of a substrate
analogue-bound P450 may be seen in Figure . The
presence of the water is thought to be the primary cause of the
stabilisation of the LS state [123]. The redox potentials of
the substrate analogue-bound complexes fall between those of the
substrate-free and substrate-bound systems. Indeed, a linear free
energy relationship between the redox potential and spin state has
been found [124].
Figure: The
active site of camphane-bound , an example of a
substrate analogue-bound system. Note that a water molecule (which is
represented by a single oxygen atom) remains coordinated with the haem
. The representation is as described in Figure .
The action of a P450 may be inhibited by many classes of molecule
[125, 126]. For example, small molecules such as CO and
NO which compete with 
in binding to the reduced haem iron
[86], metal ions (eg. 
) [125] and mechanism
based inhibitors such as chloramphenicol [127]. In this
investigation we are concerned with molecules which compete directly
with substrate molecules to bind at the active site. These ligands all
contain an electronegative atom which coordinates directly with the
haem , displacing a water molecule as the sixth axial ligand. This
is demonstrated by crystal structures of inhibitors of such as metyrapone and phenylimidazole
[98]. The UV absorption of the enzymes display a type II
shift on binding of such molecules, indicating an increase in the LS
character of the .