The importance of porphyrin distortions for the ferrochelatase reaction

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Abstract
Ferrochelatase is the terminal enzyme in haem biosynthesis, i.e. the enzyme that inserts a ferrous ion into the porphyrin ring. Suggested reaction mechanisms for this enzyme involve a distortion of the porphyrin ring when it is bound to the enzyme. We have examined the energetics of such distortions using various theoretical calculations. With the density functional B3LYP method we calculate how much energy it costs to tilt one of the pyrrole rings out of the porphyrin plane for an isolated porphyrin molecule without or with a divalent metal ion in the centre of the ring. A tilt of 30° costs 65–130 kJ/mol for most metal ions, but only ~48 kJ/mol for free-base (neutral) porphine. This indicates that once the metal is inserted, the porphyrin becomes stiffer and flatter, and therefore binds with lower affinity to a site designed to bind a distorted porphyrin. This would facilitate the release of the product from ferrochelatase. This proposal is strengthened by the fact that the only tested metal ion with a lower distortion energy than free-base porphyrin (Cd2+) is an inhibitor of ferrochelatase. Moreover, it costs even less energy to tilt a doubly deprotonated porphine2– molecule. This suggests that the protein may lower the acid constant of the pyrrole nitrogen atoms by deforming the porphyrin molecule. We have also estimated the structure of the protoporphyrin IX substrate bound to ferrochelatase using combined quantum chemical and molecular mechanics calculations. The result shows that the protein may distort the porphyrin by ~20 kJ/mol, leading to a distinctly non-planar structure. All four pyrrole rings are tilted out of the porphyrin mean plane (1–16°) but most towards the putative binding site of the metal ion. The predicted tilt is considerably smaller than that observed in the crystal structure of a porphyrin inhibitor.