Structure and mechanistic studies on the terminal step of heme biosynthesis

This part of the project is focused on understanding of the detailed mechanism of the reaction of enzyme-catalysed metallation of porphyrin. Around 10 years ago we solved the first structure of a ferrochelatase (from Bacillus subtilis), the terminal enzyme of heme biosynthesis, which catalyses the insertion of Fe²⁺ into protoporhyrin IX (Al-karadaghi et al, 1997). The structure suggested the location of the porphyrin binding cleft, while additional metal soaking experiments showed the active-site residues which were involved in metal binding. The structure created a basis for subsequent work aimed at the understanding of the mechanistic principles of the metallation reaction.

Later we managed to solve the first structure of a complex of the enzyme with a transition-state inhibitor N-methyl mesoporphyrin (Lecerof et al., 2000). This was a much awaited peace of work, since before this the research in the filed of porphyrin metallation was primarily focused on non-enzymatic metallation in organic solution. These works led to the suggestion that the enzymatic metallation should include distortion of the porphyrin substrate by an enzyme as one of the factors which contribute to the acceleration of the reaction. This distortion would expose one of the pyrrole ring nitrogen atoms to the incoming metal, thus forming the first metal-porphyrin bond. The structure of the complex clearly outlined the porphyrin binding pocket of the enzyme, revealed the amino acid residues involved in porphyrin binding and suggested the mechanistic mode of ring distortion. Moreover, by soaking Cu²⁺ into the crystals, we could show that the metal could be incorporated into the porphyrin at a very slow rate, which confirmed that the enzyme complex was in a productive mode. The nature of the active site residues involved in directing the metal into the macrocycle ring was recently confirmed (Karlberg et al., 2008) when the same complex was obtained with one of the active-site residues (His183, B. subtilis numbering), replaced by an alanine. In this case Cu²⁺-soaking did not show any metal incorporation into the porphyrin.

Another milestone in our understanding of the metallation reaction was achieved (Shipovskov et al, 2005) when by the combination of X-ray crystallography and mass-spectrometry we could show that the rate of metal insertion into N-methyl mesoporphyrin in the presence of ferrochelatase was about 20 times faster than non-enzymatic metallation. The work also suggested that acceleration of ligand exchange (protein to porphyrin) was one of the factors which contributed to the acceleration of the enzymatic metallation reaction. Based on the experimental data it was also suggested that the type of distortion of the macrocycle may contribute to the specificity of the reaction towards the metal being inserted. This hypothesis was later presented in an opinion article in Trends Biochem. Sc. (Al-Karadaghi et al., 2006). This paper summarises the view on the mechanism of the ferrochelatase reaction which emerged from the structural data obtained by us and our collaborator Professor G Ferreira. The paper was featured on the cover of TIBS (see Figure).