Molecular characterisation of coproporphyrinogen oxidase from Glycine max and Arabidopsis thaliana

Coproporphyrinogen III oxidase (CPO; E.C. 1.3.3.3 ) is an enzyme of haem and chlorophyll synthesis. Biochemical studies have indicated that the majority of CPO activity is present in plastids, with no detectable levels in mitochondria. However, this approach cannot rule out low (less than 5%) activity in the mitochondria, nor the possible presence of CPO in the cytosol, where it is found in yeast (Saccharomyces cerevisiae). We have studied this question further using molecular techniques. A cDNA encoding the mature protein of soybean (Glycine max L.) CPO was used to overexpress the enzyme 200-fold in Escherichia coli. The recombinant enzyme, purified to homogeneity in three steps, is a dimer, with a Km for coproporphyrinogen III of 0.25 ± 0.03 μM and a Vmax of 1.48 pkat. Antibodies raised against the purified soybean CPO were used in western blots to show that the enzyme is present in etioplasts but not in mitochondria. In the completely sequenced genome of Arabidopsis thaliana, we identified two genes encoding CPO, but only one of them (AtCPO-I ) was able to complement a yeast mutant defective in the enzyme; the other is likely to be a pseudogene. A construct encoding the first 92 residues of AtCPO-I fused to green fluorescent protein (GFP) was introduced into Arabidopsis plants by Agrobacterium-mediated transformation. Confocal microscopy demonstrated that the CPO–GFP fusion protein was confined exclusively to plastids in leaves and roots, with no GFP seen in the mitochondria or cytosol.