R. Franco et al., CHARACTERIZATION OF THE IRON-BINDING SITE IN MAMMALIAN FERROCHELATASEBY KINETIC AND MOSSBAUER METHODS, The Journal of biological chemistry, 270(44), 1995, pp. 26352-26357
All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.
1) to catalyze the terminal step of the heme biosynthetic pathway, whi
ch involves the insertion of ferrous ion into protoporphyrin IX. Kinet
ic methods and Mossbauer spectroscopy have been used in an effort to c
haracterize the ferrous ion-binding active site of recombinant murine
ferrochelatase. The kinetic studies indicate that dithiothreitol, a re
ducing agent commonly used in ferrochelatase activity assays, interfer
es with the enzymatic production of heme. Ferrochelatase specific acti
vity values determined under strictly anaerobic conditions are much gr
eater than those obtained for the same enzyme under aerobic conditions
and in the presence of dithiothreitol. Mossbauer spectroscopy conclus
ively demonstrates that, under the commonly used assay conditions, dit
hiothreitol chelates ferrous ion and hence competes with the enzyme fo
r binding the ferrous substrate. Mossbauer spectroscopy of ferrous ion
incubated with ferrochelatase in the absence of dithiothreitol shows
a somewhat broad quadrupole doublet. Spectral analysis indicates that
when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelmin
g majority of the added ferrous ion is bound to the protein. The spect
roscopic parameters for this bound species are delta = 1.36 +/- 0.03 m
m/s and Delta E(Q) = 3.04 +/- 0.06 mm/s, distinct from the larger Delt
a E(Q) of a control sample of Fe(II) in buffer only. The parameters fo
r the bound species are consistent with an active site composed of nit
rogenous/oxygenous Ligands and inconsistent with the presence of sulfu
r ligands. This finding is in accord with the absence of conserved cys
teines among the known ferrochelatase sequences. The implications thes
e results have with regard to the mechanism of ferrochelatase activity
are discussed.