Improved resolution of 3-mercaptopropionate dioxygenase active site provided by ENDOR spectroscopy offers insight into catalytic mechanism

Pierce, Brad S.; Schmittou, Allison N.; York, Nicholas J.; Madigan, Ryan P.; Nino, Paula F.; Foss, Frank W.; Lockart, Molly M.

doi:10.1016/j.jbc.2024.105777

Cited by 1 publication

(1 citation statement)

References 84 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Two proposed mechanistic pathways emerge from the extensive characterization of the founding member of the thiol dioxygenase superfamily, cysteine dioxygenase (CDO), and the synthetic non-heme Fe model complexes. − O 2 , a diradical molecule, ligates to the substrate-bound Fe(II) center, forming an Fe(III)-bound single radical in the form of superoxo, marking the divergence of the two pathways (Scheme ). The pathway illustrated at the top involves non-ferryl-dependent concurrent dioxygen transfer to the thiolate group of the bound l -cysteine substrate.…”

Section: Introductionmentioning

confidence: 99%

Cobalt(II)-Substituted Cysteamine Dioxygenase Oxygenation Proceeds through a Cobalt(III)-Superoxo Complex

Li,

Duan,

Liu

2024

J. Am. Chem. Soc.

View full text Add to dashboard Cite

We investigated the metal-substituted catalytic activity of human cysteamine dioxygenase (ADO), an enzyme pivotal in regulating thiol metabolism and contributing to oxygen homeostasis. Our findings demonstrate the catalytic competence of cobalt(II)-and nickel(II)-substituted ADO in cysteamine oxygenation. Notably, Co(II)-ADO exhibited superiority over Ni(II)-ADO despite remaining significantly less active than the natural enzyme. Structural analyses through X-ray crystallography and cobalt K-edge excitation confirmed successful metal substitution with minimal structural perturbations. This provided a robust structural basis, supporting a conserved catalytic mechanism tailored to distinct metal centers. This finding challenges the proposed high-valent ferryl-based mechanism for thiol dioxygenases, suggesting a non-high-valent catalytic pathway in the native enzyme. Further investigation of the cysteamine-bound or a peptide mimic of N-terminus RGS5 bound Co(II)-ADO binary complex revealed the metal center's high-spin (S = 3/2) state. Upon reaction with O 2 , a kinetically and spectroscopically detectable intermediate emerged with a ground spin state of S = 1/2. This intermediate exhibits a characteristic 59 Co hyperfine splitting (A = 67 MHz) structure in the EPR spectrum alongside UV−vis features, consistent with known low-spin Co(III)-superoxo complexes. This observation, unique for protein-bound thiolate-ligated cobalt centers in a protein, unveils the capacities for O 2 activation in such metal environments. These findings provide valuable insights into the nonheme iron-dependent thiol dioxygenase mechanistic landscape, furthering our understanding of thiol metabolism regulation. The exploration of metal-substituted ADO sheds light on the intricate interplay between metal and catalytic activity in this essential enzyme.

show abstract