title: Second-sphere interactions between the C93-Y157 cross-link and the substrate-bound Fe site influence the O₂ coupling efficiency in mouse cysteine dioxygenase.
abstract: 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 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.
authors.affiliation: Department of Chemistry and Biochemistry, College of Sciences, The University of Texas at Arlington , Arlington, Texas 76019, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Blaesi
authors.forename: Michael D
authors.initials: MD
authors.lastname: Pecore
authors.forename: Joshua K
authors.initials: JK
authors.lastname: Crowell
authors.forename: Brad S
authors.initials: BS
authors.lastname: Pierce
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D050558:Cysteine Dioxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D004791:Enzyme Inhibitors; D020035:Holoenzymes; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D007501:Iron; D050558:Cysteine Dioxygenase; D003545:Cysteine; D010100:Oxygen; C013461:cysteine sulfinic acid