The 3-His Metal Coordination Site Promotes the Coupling of Oxygen Activation to Cysteine Oxidation in Cysteine Dioxygenase.

Cysteine 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.

Not as easy as π: An insertional residue does not explain the π-helix gain-of-function in two-component FMN reductases.

The π-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The π-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4-helix. Variants of Tyr118 were generated, and their X-ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π-helix. The structure of the Y118A SsuE π-helix was converted to an α-helix, similar to the FMN-bound members of the NADPH:FMN reductase family. Although the π-helix was altered, the FMN binding region remained unchanged. Conversely, deletion of Tyr118 disrupted the secondary structural properties of the π-helix, generating a random coil region in the middle of helix 4. Both the Y118A and Δ118 SsuE SsuE variants crystallize as a dimer. The MsuE FMN reductase involved in the desulfonation of methanesulfonates is structurally similar to SsuE, but the π-helix contains a His insertional residue. Exchanging the π-helix insertional residue of each enzyme did not result in equivalent kinetic properties. Structure-based sequence analysis further demonstrated the presence of a similar Tyr residue in an FMN-bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π-helix. Results from the structural and functional studies of the FMN-dependent reductases suggest that the insertional residue alone is not solely responsible for generating the π-helix, and additional structural adaptions occur to provide the altered gain of function.