Coproporphyrinogen oxidase (copro’gen oxidase) is an enzyme in the heme biosynthetic pathway that catalyzes the oxidative decarboxylation of two propionate sidechains on coproporphyrinogen-III to produce protoporphyrinogen-IX via the monovinyl intermediate harderoporphyrinogen.  Defects in this enzyme result in metabolic diseases known as porphyrias.  As yet, the mechanisms by which the oxygen dependent enzyme selects its substrate and then catalyzes the oxidative decarboxylations are not well understood.  The absence of X-ray crystallographic data, and the lack of information on the molecular mechanism for the oxidative decarboxylation, makes copro’gen oxidase one of the least well characterized enzymes in the heme biosynthetic pathway.  We have synthesized a series of substrate analogues as probes for the active site of copro’gen oxidase and have carried out extensive studies on their metabolism by crude enzyme preparations from chicken red blood cells, an excellent source of the active enzyme.  The metabolites have been characterized by mass spectrometry, and where possible, proton NMR spectroscopy.  Enzyme kinetics were also carried out to further assess the ability of the enzyme to process these artificial substrates.  A model has been developed to explain the results from these studies.  This requires a binding site for a second propionate moiety, and the accommodation of small nonpolar groups at a second position relative to the catalytic site.  The model also is being further tested using highly purified, cloned copro’gen oxidase.