Second-Sphere Interactions between the C93–Y157 Cross-Link and the Substrate-Bound Fe Site Influence the O2 Coupling Efficiency in Mouse Cysteine Dioxygenase
2013
Li, Wei | Blaesi, Elizabeth J. | Pecore, Michael D. | Crowell, Joshua K. | Pierce, Brad S.
Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O₂-dependent oxidation of l-cysteine (l-Cys) to produce cysteinesulfinic acid (CSA). Adjacent to the Fe site of CDO is a covalently cross-linked cysteine–tyrosine pair (C93–Y157). While several theories have been proposed for the function of the C93–Y157 pair, the role of this post-translational modification remains unclear. In this work, the steady-state kinetics and O₂/CSA coupling efficiency were measured for wild-type CDO and selected active site variants (Y157F, C93A, and H155A) to probe the influence of second-sphere enzyme–substrate interactions on catalysis. In these experiments, it was observed that both kcₐₜ and the O₂/CSA coupling efficiency were highly sensitive to the presence of the C93–Y157 cross-link and its proximity to the substrate carboxylate group. Complementary electron paramagnetic resonance (EPR) experiments were performed to obtain a more detailed understanding of the second-sphere interactions identified in O₂/CSA coupling experiments. Samples of the catalytically inactive substrate-bound Feᴵᴵᴵ–CDO species were treated with cyanide, resulting in a low-spin (S = ¹/₂) ternary complex. Remarkably, both the presence of the C93–Y157 pair and interactions with the Cys carboxylate group could be readily identified by perturbations to the rhombic EPR signal. Spectroscopically validated active site quantum mechanics/molecular mechanics and density functional theory computational models are provided to suggest a potential role for Y157 in the positioning of the substrate Cys in the active site and to verify the orientation of the g-tensor relative to the CDO Fe site molecular axis.
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