M. Gora et al., PROBING THE ACTIVE-SITE RESIDUES IN SACCHAROMYCES-CEREVISIAE FERROCHELATASE BY DIRECTED MUTAGENESIS - IN-VIVO AND IN-VITRO ANALYSES, The Journal of biological chemistry, 271(20), 1996, pp. 11810-11816
Ferrochelatase is a mitochondrial inner membrane-bound enzyme that cat
alyzes the insertion of ferrous iron into protoporphyrin, the terminal
step in protoheme biosynthesis. The functional/structural roles of 10
invariant amino acid residues were investigated by site-directed muta
genesis in the yeast Saccharomyces cerevisiae ferrochelatase. The muta
nt enzymes were expressed in a yeast strain lacking the ferrochelatase
gene HEM15 and in Escherichia coli. The kinetic parameters of the mut
ant enzymes were determined for the enzymes associated with the yeast
membranes and the enzymes in the bacterial soluble fraction, They were
compared with the in vivo functioning of the mutant enzymes. The main
conclusions are the following, Glu-314 is critical for catalysis, and
we suggest that it is the base responsible for abstracting the N-pyrr
ole proton(s). His-235 is essential for metal binding, Asp-246 and Tyr
-248 are also involved in metal binding in a synergistic manner. The K
-m for protoporphyrin was also increased in the H235L, D246A, and Y248
L mutants, suggesting that the binding sites of the two substrates are
not independent of each other. The R87A, Y95L, Q111E, Q273E, W282L, a
nd F308A mutants had 1.2-2-fold increased V-m and 4-10-fold increased
K-m values for protoporphyrin, but the amount of heme made in vivo was
10-100% of the normal value. These mutations probably affected the ge
ometry of the active center, resulting in improper positioning of prot
oporphyrin.