HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 35, 23685-23691, August 29, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/35/23685    most recent M803372200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Hunter, G. A. Articles by Ferreira, G. C. PubMed PubMed Citation Articles by Hunter, G. A. Articles by Ferreira, G. C. Social Bookmarking                      What's this?

Metal Ion Substrate Inhibition of Ferrochelatase^*

Gregory A. Hunter¹, Matthew P. Sampson, and Gloria C. Ferreira²

From the Department of Molecular Medicine, College of Medicine and the H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Robust kinetic analyses of the reaction mechanism are complicated by the instability of ferrous iron in aqueous solution, particularly at alkaline pH values. At pH 7.00 the half-life for spontaneous oxidation of ferrous ion is approximately 2 min in the absence of metal complexing additives, which is sufficient for direct comparisons of alternative metal ion substrates with iron. These analyses reveal that purified recombinant ferrochelatase from both murine and yeast sources inserts not only ferrous iron but also divalent cobalt, zinc, nickel, and copper into protoporphyrin IX to form the corresponding metalloporphyrins but with considerable mechanistic variability. Ferrous iron is the preferred metal ion substrate in terms of apparent k_cat and is also the only metal ion substrate not subject to severe substrate inhibition. Substrate inhibition occurs in the order Cu²⁺ > Zn²⁺ > Co²⁺ > Ni²⁺ and can be alleviated by the addition of metal complexing agents such as β-mercaptoethanol or imidazole to the reaction buffer. These data indicate the presence of two catalytically significant metal ion binding sites that may coordinately regulate a selective processivity for the various potential metal ion substrates.

Received for publication, May 2, 2008 , and in revised form, June 10, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM080270. This work was also supported by American Heart Association Florida Affiliate Grant 0655091B (to G. C. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.

¹ To whom correspondence may be addressed: Dept. of Molecular Medicine, College of Medicine, MDC 7, University of South Florida, Tampa, FL 33612-4799. Tel.: 813-974-6176; Fax: 813-974-5798; E-mail: ghunter{at}health.usf.edu. ² To whom correspondence may be addressed: Dept. of Molecular Medicine, College of Medicine, MDC 7, University of South Florida, Tampa, FL 33612-4799. Tel.: 813-974-5797; Fax: 813-974-0504; E-mail: gferreir{at}health.usf.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?