Crystal Structure of Coproporphyrinogen Oxidase (CPO).

CPO, an essential enzyme in the heme biosynthetic pathway, catalyzes the oxidative decarboxylation of two propionate side chains of coproporphyrinogen III to form vinyl groups in the product protoporphyrinogen IX. CPO mutations are responsible for the disease hereditary coproporphyria. All eukaryotes express a highly conserved, oxygen-dependant form of CPO. Yeast and human CPO have a sequence identity of 52% at the amino acid level. To probe the biochemical basis of catalytic activity and to determine the deleterious effects of clinically identified mutations, we determined the crystal structure of CPO from S. cerevisiae. This is the first reported structure of a eukaryotic CPO. A cDNA encoding CPO (Hem13p) was expressed in E. coli as a histidine tagged protein. Hem13p was purified and concentrated to 25 mg/ml and crystals were grown in the presence of 18% PEG 8000, 0.1 M HEPES, pH 7.5, 2% isopropanol, 0.2 M Na-acetate. The crystal structure was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 Å. The protein folds into a novel structure featuring a central flat seven-stranded antiparallel beta-sheet flanked on each side with helices. The homodimeric structure of CPO is formed by a short isolated strand that forms a beta-ladder between the two monomers. Each monomer contains an active site formed between the flat beta-sheet and adjacent helices near the dimer interface. Many of the conserved residues are located at this interface. The deep active site cleft is lined by conserved residues and has been captured in an open and a closed conformation in two different crystal forms. The substrate cavity is completely buried in the closed conformation by an approximate 8 Å movement of a helix that forms a lid over the active site. The volume of the enclosed cavity precisely accommodates a modeled molecule of coproporphyrinogen III. The model indicates binding of two propionate side chains of coproporphyrinogen with two invariant arginines. The pyrrole nitrogens of the substrate are positioned to be coordinated by an invariant asparagine in a fashion similar to that described for uroporphyrinogen decarboxylase (EMBO J, 2003, 22: 6225–33). Nineteen point mutations have been described in association with hereditary coproporphyria and we have mapped these to the crystal structure. Only two mapped to the active site. Fourteen mutations were predicted to destabilize the protein and 4 of these occur at the dimer interface. Three mutations mapped to the solvent exposed surface of CPO. The crystal structure supports a model in which the enzymatic reaction occurs in an isolated cavity formed by a conformational change induced by substrate binding. The conformational change could create a binding site for molecular oxygen, the cofactor, and provide a mechanism to protect coproporphyrinogen III from inappropriate oxidation and the subsequent peroxidation of protoporphyrinogen IX.