Structure and mechanism of iron and magnesium chelatases - at the heme-chlorophyll branch-point

Sammanfattning: Tetrapyrroles are synthesised through a single branched biosynthetic pathway. Heme and chlorophyll are synthesised from the common intermediate protoporphyrin IX at the heme-chlorophyll branch-point. Ferrochelatase catalyses the synthesis of heme by inserting Fe(II) into the protoporphyrin macro-cycle while magnesium chelatase, a three-subunit enzyme, catalyses the first unique step in chlorophyll biosynthesis by inserting Mg(II). The structure of ferrochelatase has been determined both in apo form (1.8 Å) and in complex with the inhibitor N-methyl-mesoporphyrin (1.9 Å). The structure is made up of two Rossmann type domains with an active-site cleft between them. The protein induces a distortion in the porphyrin macro-cycle on binding, exposing the pyrrolenine nitrogen atoms to the in-coming metal. Metal binding studies with substrate (Zn) and inhibitor (Cd) ions reveal the metal binding residues as being the His-Glu couple, H183 and E264. Substrate and inhibitor metal ions showed different coordination geometries at the His-Glu couple and the inhibitor was bound at an additional site. A fully hydrated Mg(II) ion is bound to a pi-helix close to the active-site cleft and interacts with the metal bound at the His-Glu couple. An improved pi-helix definition algorithm enabled detailed analysis of the occurrence, conformational features, amino acid propensities and functional relevance of the pi-helix in proteins structures. The pi-helix was found to be more abundant than previously believed, and possessed unique sequence and structural features that are functionally important, as exemplified by the Mg(II) binding site of ferrochelatase. The structure of the ATPase subunit of magnesium chelatase has been determined to 2.1 Å resolution. It belongs to the AAA+ family but possesses a unique arrangement of domains. Electron micrographs of solutions of this subunit in the presence of ATP revealed ring-like structures akin to AAA hexamers. A model is proposed for the initial magnesium chelatase complex, which involves a two-tiered hexameric ring.