Human coproporphyrinogen oxidase is not a metalloprotein

Amy E. Medlock, Harry A. Dailey

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Coproporphyrinogen oxidase (CPO) (EC 1.3.3.3), the antepenultimate enzyme in the heme biosynthetic pathway, catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen IX. Previously, based upon metal analysis and site-directed mutagenesis of purified recombinant enzyme, it has been suggested that CPO contains and requires copper for activity (Kohno, H., Furukawa, T., Tokunaga, R., Taketani, S., and Yoshinaga, T. (1996) Biochim. Biophys. Acta 1292, 156-162). To examine this putative metal site in human CPO, the cDNA encoding human CPO was engineered into an expression vector with a His6 tag at its amino terminus, and the protein was expressed in Escherichia coli and purified to apparent homogeneity using nickel- nitroliotriacetic acid resin. Activity of the purified protein was monitored by a coupled fluorometric assay that employed purified protoporphyrinogen oxidase to convert protoporphyrinogen to protoporphyrin, thereby allowing the direct fluorescent determination of protoporphyrin IX produced. CPO has an apparent K(m) of 0.6 μM and an apparent K(cat) of 16 min-1 with coproporphyrinogen III as substrate. Metal analysis of the enzyme was carried out via ultraviolet and visible spectroscopy, inductively coupled plasma atomic emission spectroscopy metal analysis, and electron paramagnetic resonance spectroscopy. The data presented demonstrate that human CPO contains no metal center, that it is not stimulated in vitro by iron or copper, and that addition of these metals to cultures expressing the protein has no effect.

Original languageEnglish (US)
Pages (from-to)32507-32510
Number of pages4
JournalJournal of Biological Chemistry
Volume271
Issue number51
DOIs
StatePublished - Dec 1 1996

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Coproporphyrinogen Oxidase
Metalloproteins
Metals
Metal analysis
His-His-His-His-His-His
Copper
Spectrum Analysis
Protoporphyrinogen Oxidase
Enzymes
Spectroscopy
Atomic emission spectroscopy
Atomic Spectrophotometry
Mutagenesis
Proteins
Biosynthetic Pathways
Electron Spin Resonance Spectroscopy
Inductively coupled plasma
Site-Directed Mutagenesis
Nickel
Heme

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Human coproporphyrinogen oxidase is not a metalloprotein. / Medlock, Amy E.; Dailey, Harry A.

In: Journal of Biological Chemistry, Vol. 271, No. 51, 01.12.1996, p. 32507-32510.

Research output: Contribution to journalArticle

Medlock, Amy E. ; Dailey, Harry A. / Human coproporphyrinogen oxidase is not a metalloprotein. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 51. pp. 32507-32510.
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