HUMAN COPROPORPHYRINOGEN OXIDASE IS NOT A METALLOPROTEIN

Coproporphyrinogen oxidase (CPO) (EC 1.3.3.3), the antepenultimate enz yme in the heme biosynthetic pathway, catalyzes the conversion of copr oporphyrinogen III to protoporphyrinogen IX, Previously, based upon me tal analysis and site-directed mutagenesis of purified recombinant enz yme, it has been suggested that CPO contains and requires copper for a ctivity (Kohno, H., Furukawa, T., Tokunaga, R., Taketani, S., and Yosh inaga, T. (1996) Biochim, Biophys, Acta 1292, 156-162), To examine thi s putative metal site in human CPO, the cDNA encoding human CPO was en gineered into an expression vector with a His(6) tag at its amino term inus, and the protein was expressed in Escherichia coil and purified t o apparent homogeneity using nickel-nitrolio-triacetic acid resin. Act ivity of the purified protein was monitored by a coupled fluorometric assay that employed purified protoporphyrinogen oxidase to convert pro toporphyrinogen to protoporphyrin, thereby allowing the direct fluores cent determination of protoporphyrin IX produced, CPO has an apparent K-m of 0.6 mu M and an apparent K-cat of 16 min(-1) with coproporphyri nogen III as substrate. Metal analysis of the enzyme was carried out v ia ultraviolet and visible spectroscopy, inductively coupled plasma at omic emission spectroscopy metal analysis, and electron paramagnetic r esonance spectroscopy, The data presented demonstrate that human CPO c ontains 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.