Ferrochelatase and Magnesiumchelatase: Metal chelation studied with mutants

Abstract: Magnesium chelatase and ferrochelatase are two very important enzymes, which are involved in chlorophyll and heme biosynthesis, respectively. They both use protoporphyrin IX as substrate but magnesium chelatase inserts a magnesium ion whereas ferrochelatase inserts a ferrous ion. These two enzymes have been studied in this thesis. Even though they both insert a metal ion into protoporphyrin IX they are very different. Magnesium chelatase is a multimeric enzyme comprised of three different subunits that requires ATP for chelation while ferrochelatase in a monomeric enzyme that does not need ATP for chelation to occur. Ferrochelatase is relatively well characterised and the aim of the research has been to explain the mechanism of the chelation reaction, while the research on magnesium chelatase that is not that well known has been focused on basic characterisation. This thesis is based on six papers. Paper I suggests an amino acid residue located on the outside of the ferrochelatase as being a site for interaction with another protein. This protein is suggested to deliver substrate or retrieve product. Paper II investigated the amino acids residues, which are involved in the binding of the metal substrate to ferrochelatase. Paper III investigates the interaction between BchI and BchD subunits of magnesium chelatase. Paper IV is a characterisation at DNA, mRNA and protein level of barley xantha-f mutants. The gene product is the largest subunit of the magnesium chelatase. Paper V investigates conserved amino acid residues responsible for the binding of the protoporphyrin IX substrate to the largest subunit of magnesium chelatase and estimate Kd values for deuteroporphyrin IX binding to BchH. The results of the binding studies indicate the presence of two types of binding site to the BchH subunit. Paper VI demonstrates so-called gun (genomes uncoupled) phenotype in barley xantha-f, -g and –h mutants, but not in a xantha-l mutants. The study supports Mg-protoporphyrin IX as a signal molecule in chloroplast-to-nucleus signal transduction.