CHARACTERIZATION OF THE IRON-BINDING SITE IN MAMMALIAN FERROCHELATASEBY KINETIC AND MOSSBAUER METHODS

Citation
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
Citations number
42
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
270
Issue
44
Year of publication
1995
Pages
26352 - 26357
Database
ISI
SICI code
0021-9258(1995)270:44<26352:COTISI>2.0.ZU;2-6
Abstract
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.