In order to investigate the rôle played by the water molecule which
is coordinated with the haem 
ion in the ligand-free and
substrate analogue-bound complexes, a series of computational
experiments were performed. The water molecule was removed from those
systems in which it is naturally found and, in the camphor-bound case, a
water molecule was inserted in the same position
relative to the ion as found in the substrate analogue
complexes. The remaining atomic positions were unchanged, allowing the
direct effect of the presence of the water to be isolated. The results
of the ab initio simulations of the natural and modified systems
are summarised in Figure 
.
Figure: Schematic
diagrams of the 
systems modelled and the results of
calculations of the spin and charge on the in each system with
and without an -coordinated water molecule. The systems are shown
in their natural state with a fragment of the ligand if
present. Distances are given in Å, spins in 
and charges in
e. Carbon atoms are shown in grey, oxygen in red, nitrogen in light
blue, sulphur in yellow, iron in dark blue and hydrogen in white.
In the ligand-free system we can see that the removal of the
-coordinated water molecule leads to a change from a LS to a HS
state. Conversely, the spin on the changes from a high to a low
value on the addition of a water molecule to the substrate-bound
system. However, surprisingly, removing the -coordinated water from
the natural structure of either of the substrate analogue-bound
systems, leaving all other aspects of the geometry unchanged, makes no
significant difference to the spin. This indicates that in these
substrate analogue complexes there is another mechanism maintaining
the LS character of the ion.
If we examine the geometry of the haem moieties in the different systems,
shown in Figure , we can see that the binding of a
ligand causes a distortion of the haem, with the ion moving out of
the plane of the haem by between 0.19 Å and 0.25 Å. However, in
the substrate analogue-bound systems this is accompanied by the
shortening of the Fe-S bond by approximately 0.4 Å. It is possible
that the shortening of this bond causes an increase in the ligand
field on the ion which stabilises the LS state, despite the fact
that the is only five-fold coordinated. In order to test
this hypothesis, a series of simulations were performed in which the
Fe-S bond lengths were increased to 2.4 Å in both of the substrate
analogue complexes, leaving the remaining geometry unchanged. The
results of the calculations of the spin and charge with and
without an -coordinated water are summarised in Figure
. From these results we can see that the
spin state changes from LS to HS in both systems on the removal of
the water molecule, as we would expect when changing from six- to
five-fold coordination . The bond populations between the Fe and S
atoms may also be calculated. These show that the overlap population
decreases from 0.56e to 0.46e on extending the Fe-S bond in the
camphane-bound system and from 0.47e to 0.41e in the
norcamphor-bound complex 
. This
indicates that the shortening of the bond causes an increase in the
bonding interaction between the Fe and S atoms. Thus we can conclude
that the shortening of the bond between the haem iron and cysteine
sulphur is responsible for maintaining the LS character of the ion
in these systems.
Figure: Schematic diagrams of the modified substrate analogue-bound
complexes of and the results of calculations of the spin and
charge on the with and without an -coordinated water
molecule. The Fe-S bond has been stretched to 2.4Å in each
case. The representation is as described in Figure .
The most probable cause of the decrease in the length of the Fe-S
bond may be seen if we examine the charge on the ion in each of
the complexes. In the ligand-bound systems, in which the ion has
moved out of the plane of the haem, we can see that the presence of
an -coordinated water molecule causes an increase in the charge
by approximately 0.1e. As the cysteine S atom is negatively charged,
this will cause an increase in the attraction between these
ions. Therefore, we see that the presence of the water in these
substrate analogue-bound systems probably leads to the energetic
favourability of the LS state of the , but that the LS state of the
is not a direct effect of the presence of the water as postulated
in the standard model of the interaction.
