Conformation of the intermediates in the reaction catalyzed by protoporphyrinogen oxidase: An in silico analysis

Abstract

Protoporphyrinogen oxidase (PPO) is a critical enzyme across life as the last common step in the synthesis of many metalloporphyrins. The reaction mechanism of PPO was assessed in silico and the unstructured loop near the binding pocket was investigated. The substrate, intermediates, and product were docked in the catalytic domain of PPO using a modified Autodock method, introducing flexibility in the macrocycles. Sixteen PPO protein sequences across phyla were aligned and analyzed with Phyre2 and ProteinPredict to study the unstructured loop from residue 204–210 in the H. sapiens structure. Docking of the substrate, intermediates, and product all resulted in negative binding energies, though the substrate had a lower energy than the others by 40%. The α-H of C10 was found to be 1.4 angstroms closer to FAD than the β-H, explaining previous reports of the reaction occurring on the meso face of the substrate. A lack of homology in sequence or length in the unstructured loop indicates a lack of function for the protein reaction. This docking study supports a reaction mechanism proposed previously whereby all hydride abstractions occur on the C10 of the tetrapyrrole followed by tautomeric rearrangement to prepare the intermediate for the next reaction.