Organic fluorochemicals are widely distributed in the environment, causing ecological and health concerns. However, defluorination is a challenging process. This article summarizes the defluorination mechanisms learned from metal-containing enzymes.

Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a non-heme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2. Here, using EPR, Mössbauer, and UV-Vis spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate to the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed a substrate-mediated reduction of the ferric to the ferrous oxidation state at the iron center. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases.

Cysteamine dioxygenase (ADO) plays a vital role in regulating thiol metabolism and preserving oxygen homeostasis in humans by oxidizing the sulfur of cysteamine and N-terminal cysteine-containing proteins to their corresponding sulfinic acids using O₂ as a cosubstrate. However, as the only thiol dioxygenase that processes both small-molecule and protein substrates, how ADO handles diverse substrates of disparate sizes to achieve various reactions is not understood. The knowledge gap is mainly due to the three-dimensional structure not being solved, as ADO cannot be directly compared with other known thiol dioxygenases. Herein, we report the first crystal structure of human ADO at a resolution of 1.78 Å with a nickel-bound metal center. Crystallization was achieved through both metal substitution and C18S/C239S double mutations. The metal center resides in a tunnel close to an entry site flanked by loops. While ADO appears to use extensive flexibility to handle substrates of different sizes, it also employs proline and proline pairs to maintain the core protein structure and to retain the residues critical for catalysis in place. This feature distinguishes ADO from thiol dioxygenases that only oxidize small-molecule substrates, possibly explaining its divergent substrate specificity. Our findings also elucidate the structural basis for ADO functioning as an oxygen sensor by modifying N-degron substrates to transduce responses to hypoxia. Thus, this work fills a gap in structure–function relationships of the thiol dioxygenase family and provides a platform for further mechanistic investigation and therapeutic intervention targeting impaired oxygen sensing.

LmbB2 is a peroxygenase-like enzyme that hydroxylates l-tyrosine to l-3,4-dihydroxyphenylalanine (DOPA) in the presence of hydrogen peroxide. However, its heme cofactor is ligated by a proximal histidine, not cysteine. We show that LmbB2 can oxidize l-tyrosine analogues with ring-deactivated substituents such as 3-nitro-, 3-fluoro-, 3-chloro-, and 3-iodo-l-tyrosine. We also found that the 4-hydroxyl group of the substrate is essential for reacting with the heme-based oxidant and activating the aromatic C–H bond. The most interesting observation of this study was obtained with 3-fluoro-l-tyrosine as a substrate and mechanistic probe. The LmbB2-mediated catalytic reaction yielded two hydroxylated products with comparable populations: i.e., oxidative C–H bond cleavage at C5 to generate 3-fluoro-5-hydroxyl-l-tyrosine and oxygenation at C3 concomitant with a carbon–fluorine bond cleavage to yield DOPA and fluoride. An iron protein mediated hydroxylation on both C–H and C–F bonds with multiple turnovers is unprecedented. Thus, this finding reveals a significant potential of biocatalysis in C–H/C–X bond (X = halogen) cleavage. Further ¹⁸O-labeling results suggest that the source of oxygen for hydroxylation is a peroxide and that a commonly expected oxidation by a high-valent iron intermediate followed by hydrolysis is not supported for the C–F bond cleavage. Instead, the C–F bond cleavage is proposed to be initiated by a nucleophilic aromatic substitution mediated by the iron hydroperoxo species. On the basis of the experimental results, two mechanisms are proposed to explain how LmbB2 hydroxylates the substrate and cleaves C–H/C–F bonds. This study broadens the understanding of heme enzyme catalysis and sheds light on enzymatic applications in medicinal and environmental fields.