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.