Structural Insight Into Human Protoporphyrinogen Oxidase

Protoporphyrinogen IX oxidase (PPO; EC1.3.3.4), is the last common enzyme for the transformation of protoporphyrinogen IX to protoporphyrin IX, and plays an important part in the biosynthesis of heme and chlorophyll.The inhibition of PPO leads to the accumulation of protoporphyrinogen IX, which is then exported to the cytoplasm and oxidized to protoporphyrin IX spontaneously by oxygen. In the presence of light, the photosensitizing protoporphyrin IX can further generate singlet oxygen, which induces rapid lipid peroxidation and cell death. These special characteristics of PPO enzymes underlie their role in an inherited human disease called variegated prophyria (VP) and also make it an excellent target for herbicides.In this study, we report the crystal structure of Human protoporphyrinogen IX oxidase (hPPO) in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of1.9A.The structural and biochemical analyses revealed the molecular details of FAD and acifluorfen binding to hPPO as well as the interactions of the substrate with hPPO. Structural analysis and gel chromatography indicated that hPPO is a monomer rather than a homodimer in vitro. The founder-effect mutation R59W in VP patients is most likely caused by a severe electrostatic hindrance in the hydrophilic binding pocket involving the bulky, hydrophobic indolyl ring of the tryptophan. Forty-seven VP-causing mutations were purified by chromatography and kinetically characterized in vitro. The effect of each mutation was demonstrated in the high-resolution crystal structure.The human heme biosynthesis pathway consists of8enzymes. A deficiency in each of the last7enzymes corresponds with a variant of porphyria. The crystal structure of each these enzymes has been determined, except for hPPO. Here, with the current hPPO structure, we have all7structures at hand. We believe that these structures will provide important information not only for understanding the heme biosynthesis but also for further elucidating the molecular mechanism of porphyria.