<?xml version="1.0" encoding="UTF-8"?><xml><records><record><source-app name="Biblio" version="6.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors><authors><author><style face="normal" font="default" size="100%">Franco, R.</style></author><author><style face="normal" font="default" size="100%">Moura, J. J.</style></author><author><style face="normal" font="default" size="100%">Moura, I.</style></author><author><style face="normal" font="default" size="100%">Lloyd, S. G.</style></author><author><style face="normal" font="default" size="100%">Huynh, B. H.</style></author><author><style face="normal" font="default" size="100%">Forbes, W. S.</style></author><author><style face="normal" font="default" size="100%">Ferreira, G. C.</style></author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mossbauer methods</style></title><secondary-title><style face="normal" font="default" size="100%">J Biol Chem</style></secondary-title></titles><keywords><keyword><style  face="normal" font="default" size="100%">Animals</style></keyword><keyword><style  face="normal" font="default" size="100%">Binding Sites</style></keyword><keyword><style  face="normal" font="default" size="100%">Cloning, Molecular</style></keyword><keyword><style  face="normal" font="default" size="100%">Dithiothreitol/metabolism</style></keyword><keyword><style  face="normal" font="default" size="100%">Escherichia coli</style></keyword><keyword><style  face="normal" font="default" size="100%">Ferrochelatase/*chemistry/isolation &amp; purification/*metabolism</style></keyword><keyword><style  face="normal" font="default" size="100%">Hydrogen-Ion Concentration</style></keyword><keyword><style  face="normal" font="default" size="100%">Iron/*metabolism</style></keyword><keyword><style  face="normal" font="default" size="100%">Kinetics</style></keyword><keyword><style  face="normal" font="default" size="100%">Liver/enzymology</style></keyword><keyword><style  face="normal" font="default" size="100%">Mammals</style></keyword><keyword><style  face="normal" font="default" size="100%">Mathematics</style></keyword><keyword><style  face="normal" font="default" size="100%">Mice</style></keyword><keyword><style  face="normal" font="default" size="100%">Models, Theoretical</style></keyword><keyword><style  face="normal" font="default" size="100%">Protoporphyrins/pharmacology</style></keyword><keyword><style  face="normal" font="default" size="100%">Recombinant Proteins/chemistry/isolation &amp; purification/metabolism</style></keyword><keyword><style  face="normal" font="default" size="100%">Spectroscopy, Mossbauer/methods</style></keyword></keywords><dates><year><style  face="normal" font="default" size="100%">1995</style></year><pub-dates><date><style  face="normal" font="default" size="100%">Nov 3</style></date></pub-dates></dates><urls><web-urls><url><style face="normal" font="default" size="100%">http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&amp;db=PubMed&amp;dopt=Citation&amp;list_uids=7592847 </style></url></web-urls></urls><number><style face="normal" font="default" size="100%">44</style></number><volume><style face="normal" font="default" size="100%">270</style></volume><pages><style face="normal" font="default" size="100%">26352-7</style></pages><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">&lt;p&gt;All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX. Kinetic methods and Mossbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mossbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for 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 overwhelming majority of the added ferrous ion is bound to the protein. The spectroscopic parameters for this bound species are delta = 1.36 +/- 0.03 mm/s and delta EQ = 3.04 +/- 0.06 mm/s, distinct from the larger delta EQ of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with regard to the mechanism of ferrochelatase activity are discussed.&lt;/p&gt;
</style></abstract><accession-num><style face="normal" font="default" size="100%">7592847</style></accession-num><notes><style face="normal" font="default" size="100%">&lt;p&gt;0021-9258 (Print)0021-9258 (Linking)Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.&lt;/p&gt;
</style></notes><auth-address><style face="normal" font="default" size="100%">Departamento de Quimica, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Monte de Caparica, Portugal.</style></auth-address></record></records></xml>