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 [91]
(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 [116] 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
[117] 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 [86].
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 [118], NMR [119] and
crystallographic [93] 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
[96] 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
[76].
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 [120]. 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.
