title: Human coproporphyrinogen oxidase is not a metalloprotein.
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 Km of 0.6 microM and an apparent Kcat 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-2605, USA. Dailey@bscr.uga.edu
authors.forename: A E
authors.initials: AE
authors.lastname: Medlock
authors.forename: H A
authors.initials: HA
authors.lastname: Dailey
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003300:Copper / Q000032:analysis; D003304:Coproporphyrinogen Oxidase / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D013057:Spectrum Analysis
chemical_list: D011994:Recombinant Proteins; D003300:Copper; D007501:Iron; D003304:Coproporphyrinogen Oxidase