Mechanism of Organophosphonate Catabolism by Diiron Oxygenase PhnZ: A Third Iron-Mediated O-O Activation Scenario in Nature

Diiron oxygenase PhnZ catalyzes the catabolism of organophosphonate (Pn) (R)-2-amino-1-hydroxyethylphos-phonic to glycine and inorganic phosphate (Pi). In this Pn catabolism way, PhnZ oxidatively cleaves the highly stable C-P bond in Pn to produce Pi. However, the mechanism of this enzyme that affords aquatic and marine bacteria in Pi-limited environments to utilize the most abundant environmental Pn (2-amino-ethylphosphonic acid) as the source of Pi is still unclear. In this work, extensive QM/MM calculations reveal that the mechanism of PhnZ consists of four consecutive steps: (1) rate-limiting alpha-H abstraction of Pn by Fe-III-superoxo; (2) formation of (FeOOC alpha)-O-III peroxide; (3) concerted O insertion into C-alpha-P bond of Pn initiated by inverse heterolytic O-O cleavage; and (4) phosphate hydrolysis to glycine and Pi. Intriguingly, the enzymatic reaction mechanism of PhnZ for the crucial breakage of the C-P bond is characterized by the inverse heterolytic O-O cleavage of (FeOOC alpha)-O-III intermediate, which renders the distal O atom more oxidative to oxygenate Pn than the homolytic O-O cleavage. In this way, PhnZ adopts a mechanism quite different from the related diiron oxygenase MIOX, with His62 residue playing an important role. This unusual inverse heterolytic O-O cleavage mode, apart from the well-known homolytic and normal heterolytic ones, constitutes a third iron-mediated O-O activation scenario in nature, which is expected to have its broad occurrence in oxidative transformation involving heteroatoms of sulfur and phosphorus.