To obtain recombinant human coproporphyrinogen oxidase (CPX), a cDNA for the coding region of mature human CPX has been expressed in E. coli. CPX was produced as a fusion protein with glutathione S-transferase followed by the hexapeptide recognition site for thrombin cleavage just preceding first amino acid of the CPX protein. The human CPX was found to be in the soluble fraction. This previously unobtainable human heme synthetic enzyme was purified to electrophoretic homogeneity with a specific activity of 4200 nmol/hr./mg of protein using a Glutathione Sepharose 4B column and gel filtration. Recombinant human CPX exhibits homogeneous behavior during high performance liquid chromatography (HPLC) and the N-terminal sequence, confirmed by protein sequencing, revealed a single polypeptide chain. In its active form, human CPX is a homodimer. According to the hydrodynamic properties derived from analytical ultracentrifugation, dimeric CPX has a nearly globular shape. Additionally, naturally occurring Arg to Trp (R231W)-mutated CPX has been also expressed in E. coli and further characterized. The mutated enzyme has a Km value of 0.55 microM as compared to 0.30 microM for the wild type. The catalytic efficiency (specificity constant, kcat/Km) of the mutated CPX was four fold lower than wild-type enzyme. The activity measurement of the mutated enzyme showed higher thermal sensitivity as compared with wild type CPX. The measured pI for mutated CPX is 5.65, compared to 6.40 for wild type. The pH optima for the mutated and wild-type protein are 6.6 and 6.8, respectively. The R231W mutation of CPX does not affect dimer formation and both normal and mutated CPX exhibit identical sedimentation properties. The thermal denaturation of both wild type and mutant CPX was found to be irreversible. The mutated CPX contained a significant amount of tightly bound porphyrin coproporphyrin. No metal association was found either in wild type or in mutated CPX. The availability of the recombinant human CPX will aid in structural and mechanistic studies.