Crystallographic studies on porphyrin metallation by ferrochelatase

Sammanfattning: Ferrochelatase catalyses the terminal step in heme biosynthesis by inserting a ferrous ion into protoporphyrin IX. The reaction mechanism has in this thesis been studied by mainly crystallographic methods. The structure of ferrochelatase from Bacillus subtilis co-crystallized with N-methyl mesoporphyrin allowed for the first time a characterization of the active site of the enzyme. The structure suggests a mechanism of porphyrin distortion where the pyrrole rings B, C and D are fixed by the enzyme, while pyrrole ring A is forced to tilt 36?. Soakings of Zn2+ into ferrochelatase crystals showed that the metal was coordinated to the invariant residues His183 and Glu264 located in the active site. These residues are proposed to participate in a sitting-atop complex with the metal and porphyrin. A fully hydrated Mg2+ complex was found located 7 Å away from the Zn2+ ion. Mg2+ had a stimulatory effect on the enzyme activity. From biochemical and theoretical studies it was found that the two metals interact with each other, resulting in lower binding affinity for Zn2+. The structure of ferrochelatase from Saccharomyces cerevisiae was determined to a resolution of 2.4 Å. It was found to be a homodimer. Metal soakings with a substrate metal (Co2+) and an inhibitor (Cd2+) showed that they were bound to the same site as was observed earlier for ferrochelatase from B. subtilis. The observation that the two monomers bind metal differently suggest that porphyrin metallation in the yeast monomers occur in an alternating fashion, like a two-stroke engine.