bi9b01085_si_001.pdf (224.53 kB)
The 3‑His Metal Coordination Site Promotes the Coupling of Oxygen Activation to Cysteine Oxidation in Cysteine Dioxygenase
journal contribution
posted on 2020-05-19, 17:09 authored by Dianna
L. Forbes, Kathleen M. Meneely, Annemarie S. Chilton, Audrey L. Lamb, Holly R. EllisCysteine
dioxygenase (CDO) structurally resembles cupin enzymes
that use a 3-His/1-Glu coordination scheme. However, the glutamate
ligand is substituted with a cysteine (Cys93) residue, which forms
a thioether bond with tyrosine (Tyr157) under physiological conditions.
The reversion variant, C93E CDO, was generated in order to reestablish
the more common 3-His/1-Glu metal ligands of the cupin superfamily.
This variant provides a framework for testing the structural and functional
significance of Cys93 and the cross-link in CDO. Although dioxygen
consumption was observed with C93E CDO, it was not coupled with l-cysteine oxidation. Substrate analogues (d-cysteine,
cysteamine, and 3-mercaptopropionate) were not viable substrates for
the C93E CDO variant, although they showed variable coordinations
to the iron center. The structures of C93E and cross-linked and non-cross-linked
wild-type CDO were solved by X-ray crystallography to 1.91, 2.49,
and 2.30 Å, respectively. The C93E CDO variant had similar overall
structural properties compared to cross-linked CDO; however, the iron
was coordinated by a 3-His/1-Glu geometry, leaving only two coordination
sites available for dioxygen and bidentate l-cysteine binding.
The hydroxyl group of Tyr157 shifted in both non-cross-linked and
C93E CDO, and this displacement prevented the residue from participating
in substrate stabilization. Based on these results, the divergence
of the metal center of cysteine dioxygenase from the 3-His/1-Glu geometry
seen with many cupin enzymes was essential for effective substrate
binding. The substitution of Glu with Cys in CDO allows for a third
coordination site on the iron for bidentate cysteine and monodentate
oxygen binding.