ISOLATION AND FUNCTIONAL-CHARACTERIZATION OF MUTANT FERROCHELATASES IN SACCHAROMYCES-CEREVISIAE

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that cat alyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. It is encoded by the HEM15 gene in th e yeast Saccharomyces cerevisiae. Five hem15 mutants causing defective heme synthesis and protoporphyrin accumulation were investigated. The mutations were identified by sequencing the mutant hem15 alleles ampl ified in vitro from mutant genomic DNA. A single nucleotide change, ca using an amino acid substitution, was found in each mutant. The substi tution L62F caused a five-fold increase in V-max and 32-fold and four- fold increases in the K-M's for protoporphyrin and metal. Replacements of the conserved G47 by S and S102 by F increased the K-M for protopo rphyrin 10-fold without affecting the affinity for metal or enzyme act ivity. Two amino acid changes, L205P and P221L, produced a thermosensi tive phenotype. In vivo heme synthesis, the amount of immunodetected p rotein, and ferrochelatase activity measured in vitro were more affect ed in cells grown at 37 degrees C than at 30 degrees C. The effects of these mutations on the enzyme function are discussed with respects to ferrochelatase structure and mechanism of action.