Iron in Haem Biosynthesis. Structural Studies of Ferrochelatase and Frataxin

Sammanfattning: Ferrochelatase catalyses the synthesis of heme by inserting Fe(II) into protoporphyrin IX. The structure of ferrochelatase from yeast has been determined (2.4 Å). The structure is homodimeric with each of the monomer made up of two Rossmann type domains with an active-site cleft between them. Metal binding studies with Co(II) and Cd(II) ions reveal the metal binding residues as being the His-Glu couple, His235 and Glu314. In addition Cd(II) binds to a second site coordinating Glu97, His317 and Glu326.Metal binding studies on the bacterial ferrochelatase from Bacillus subtilis revealed an Fe(II) bound to the same His-Glu couple, His183 and Glu264. The structure was refined to 1.7 Å. Site-directed mutants in the active site were studied and four of them were structurally determined, Tyr13Phe (2.4 Å), Lys87Ala (1.2 Å), His183Cys (2.1 Å) and His183Ala (2.4 Å). In combination with biochemical studies a likely mechanism of metal binding is suggested to involve the His-Glu couple. The porphyrin inhibitor N-MeMP binds with high affinity to ferrochelatase. Cu(II) insertion into N-MeMP was studied by a time series of soaking N-MeMP:ferrochelatase crystals. Cu(II) was seen accumulating into the ring while density for the N-methyl group was seen decreasing. Mass spectrometry studies on porphyrin metallation revealed that ferrochelatase catalyse metal insertion. Cu(II) was more efficiently inserted than Zn(II) which contrasts with studies on the substrate protoporphyrin IX. The most probable reason for the discrepancy is the degree of distortion of the macro-cycle, which therefore could be of importance in metal selectivity. Co-crystal structures of two different inhibitors were determined. N-MeMP crystallised with His183Ala variant of ferrochelatase revealed a different isomer, from the racemic mixture, located in the binding cleft compared to previously determined wild-type structure. The structure was refined to 2.4 Å. His183 is put forward as an important residue controlling binding specificity. The other inihibitor, 2,4-disulphonic deuteroporphyrin IX, has two bulky side-chains and was seen trapped on its way into the binding cleft of the His183Cys ferrochelatase variant (2.1 Å). Frataxin is a mitochondrial protein involved in iron delivery and storage. The crystal structure of trimeric frataxin from yeast was determined to 3.0 Å. The structure has a propeller-like shape with the monomers making up the wings. Each monomer is folded as an alpha/beta sandwich. The same fold was also found in other metallochaperones. In an EM-reconstruction of the 24-meric oligomer the trimers were located at the corners with octahedral symmetry. Eight trimer structures could be docked into the envelope. Frataxin seems to be more dynamic in terms of its bi-functionality and iron storage properties compared to ferritin.