The cytochromes P450s are hemoproteins [82, 83], made up of between 400 and amino acids and containing a single haem prosthetic group. The distal axial ligand of this haem moiety is formed by a cysteine residue  (See Figure ).
Figure: A representation of with bound camphor. The enlarged active site region shows the camphor substrate, haem moiety and cysteine residue which forms the distal haem ligand. In the representation of the full enzyme the protein backbone is shown in green, the haem moiety in blue and the substrate is coloured according to atomic species. Oxygen atoms are shown in red, carbon in grey, nitrogen in light blue, sulphur in yellow and iron in dark blue.
(Ferric iron) can exist in two spin states, , low spin (LS) in which the five 3d electrons are maximally paired, or , high spin (HS) in which the five 3d electrons are maximally unpaired. Intermediary spin states, while possible, are not normally found in biological systems. In the haem system, six-fold coordinated is generally found to be LS and five-fold coordinated is found in the HS state. This is due to the fact that the ionic radius is larger for HS than for LS  and in the HS state the moves out of the plane of the porphyrin ring as the central cavity is too small. The nature of the axial haem ligands also have an important effect on the LS-HS balance. A strong axial field will bring about a relatively large d-orbital splitting, favouring the LS state. This can be seen in practice if one compares the UV absorption spectra of CO complexes with hemoglobin (histidine axial ligand) and P450 (cysteine ligand). The axial ligand field strength due to histidine is much stronger than that due to cysteine  and correspondingly the UV absorption band due to the transition in CO-hemoglobin occurs at 420nm, while that due to CO-P450 occurs at 450nm .
Ligand-free P450 exhibits a Soret absorption maximum in a UV spectrum at approximately 420nm. This is associated with the LS state of the . Spectral , NMR  and crystallographic  data indicate that a water molecule forms a sixth axial ligand of the in the substrate-free form, thus stabilising the LS state of the ion (See Figure ).
Figure: The active site of substrate-free . Note the water molecule (which can be seen as a single oxygen atom) that forms the sixth axial ligand of the haem iron. Oxygen atoms are shown in red, nitrogen in light blue, sulphur in yellow and iron in dark blue. Carbon atoms are shown in grey as bonds only and hydrogens have been omitted from this figure for clarity.
There are three types of ligand binding which may be identified by changes in the UV spectrum. These are known as type I, type II and reverse type I (or alternatively modified type II).
Type I binding is associated with the binding of a substrate. It is identified by a reduction in the Soret absorption band at 420nm and a corresponding increase in a maximum at 390nm with an isobestic point at 407nm (See Figure ). This is indicative of a change from a LS to HS state of the ferric iron. X-ray crystallography of substrate-bound complexes of the enzyme  shows that the iron-ligated water molecule is displaced in these cases, changing the from a six-fold to a five-fold coordination state. The ion is also seen to move out of the plane of the haem. Figure shows an example of the active site of a substrate-bound P450.
Figure: The UV difference spectra obtained by the addition of a substrate or inhibitor to cytochrome P450. The black and red lines represent type I and type II difference spectra respectively. The data for this figure was obtained from .
Figure: The active site of camphor-bound , an example of a substrate-bound system. Note the absence of the water molecule which formed the sixth axial ligand of the haem iron in the substrate-free enzyme. The representation is as described in Figure .
A type II spectrum is characterised by an increase in the absorption between 425nm and 435nm and a decrease in the 390nm maximum with an isobestic point at approximately 419nm . This is correlated with an increase in the LS character of the . This spectral change is associated with the interaction of inhibitors which bind directly to the , replacing the water molecule as the sixth axial ligand [98, 99] (See Figure ).
Figure: The active site of metyrapone-bound , and example of an inhibitor-bound system. Note that the inhibitor binds directly to the haem replacing the water molecule as the sixth axial ligand. The representation is as described in Figure .
The reverse type I spectrum is a `mirror image' of the type I spectral change, with an increase in the 420nm band and a decrease in absorbance at 390nm. This is similar to a type II change, except that there is no shift in the location of the maxima. The exact nature of this interaction is not known and it may be that the ligand binds to a different site on the enzyme.
The HS and LS states are not independent, but exist in equilibrium. The differences in the absorption and extinction coefficients between the two bands at 390 and 420nm allow the equilibrium constant between the spin states and hence the fraction of HS character to be determined. The equilibrium between HS and LS is only one of a number of microequilibria which must be considered in a full description of the substrate binding reaction. Detailed analyses of these may be found in [120, 121, 122]. The equilibrium between these states may be effected by many factors such as the pH of the solution or changes in the conformation of the enzyme which alter the nature of the binding of a ligand.