| First Author | Li W | Year | 2013 |
| Journal | Biochemistry | Volume | 52 |
| Issue | 51 | Pages | 9104-19 |
| PubMed ID | 24279989 | Mgi Jnum | J:207223 |
| Mgi Id | MGI:5554955 | Doi | 10.1021/bi4010232 |
| Citation | Li W, et al. (2013) Second-sphere interactions between the C93-Y157 cross-link and the substrate-bound Fe site influence the O(2) coupling efficiency in mouse cysteine dioxygenase. Biochemistry 52(51):9104-19 |
| abstractText | Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O(2)-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 O(2)/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 O(2)/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 O(2)/CSA coupling experiments. Samples of the catalytically inactive substrate-bound Fe(III)-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. |
