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 . 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 .
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 , metal ions (eg. )  and mechanism based inhibitors such as chloramphenicol . 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 . 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 .