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Title: Cobalt(II)-Substituted Cysteamine Dioxygenase Oxygenation Proceeds through a Cobalt(III)-Superoxo Complex
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 O2, a kinetically and spectroscopically detectable intermediate emerged with a ground spin state of S = 1/2. This intermediate exhibits a characteristic 59Co 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 O2 activation in such metal environments. These findings provide valuable insights into the non-heme 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.  more » « less
Award ID(s):
2204225
PAR ID:
10525620
Author(s) / Creator(s):
; ;
Editor(s):
DeBeer, Serena
Publisher / Repository:
American Chemical Society (ACS)
Date Published:
Journal Name:
Journal of the American Chemical Society
Volume:
146
Issue:
27
ISSN:
0002-7863
Page Range / eLocation ID:
18292 to 18297
Subject(s) / Keyword(s):
Thiol dioxygenase, metal-substitution, oxygen activation, thiol-ligation, bioinorganic chemistry, mechanistic enzymology
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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