Abstract
Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a nonheme 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 electron paramagnetic resonance (EPR), Mössbauer, and UV-visible 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 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 substrate-mediated reduction of the iron center from ferric to the ferrous oxidation state. 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.
Highlights
Thiol dioxygenases are a group of nonheme, ferrous enzymes that incorporate two oxygen atoms from molecular oxygen into the thiol groups of their corresponding substrates [1]
X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy was used to monitor the change of the oxidation state of the iron center upon incubation with cysteamine or the N terminus of regulator of G signaling 5 (RGS5)
Thiol dioxygenases are a family of 3-His coordinated ferrous enzymes that activate molecular oxygen and transform thiol substrates to corresponding sulfinates
Summary
Thiol dioxygenases are a group of nonheme, ferrous enzymes that incorporate two oxygen atoms from molecular oxygen into the thiol groups of their corresponding substrates [1] Plant cysteine oxidase (PCO) promotes the N-terminal thiol dioxygenation of VII ethylene response factors in normoxia to precede arginylation, resulting in proteasomal degradation [19] This type of posttranslational modification mediated by thiol dioxygenases was reported in ADO [20]. Some structural and mechanistic similarity is generally anticipated between ADO and CDO because of the catalytic reactions they promote, the substrate-binding mode of ADO cannot be extrapolated because of intrinsic differences
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