Spectroscopic and Computational Investigation of the H155A Variant of Cysteine Dioxygenase: Geometric and Electronic Consequences of a Third-Sphere Amino Acid Substitution

Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron­(II)-dependent enzyme that utilizes molecular oxygen to catalyze the oxidation of l-cysteine (Cys) to cysteinesulfinic acid. Although the kinetic consequences of various outer-sphere amino acid substitutions have previously been assessed, the effects of these substitutions on the geometric and electronic structures of the active site remained largely unexplored. In this work, we have performed a spectroscopic and computational characterization of the H155A CDO variant, which was previously shown to display a rate of Cys oxidation ∼100-fold decreased relative to that of wild-type (WT) CDO. Magnetic circular dichroism and electron paramagnetic resonance spectroscopic data indicate that the His155 → Ala substitution has a significant effect on the electronic structure of the Cys-bound Fe­(II)­CDO active site. An analysis of these data within the framework of density functional theory calculations reveals that Cys-bound H155A Fe­(II)­CDO possesses a six-coordinate Fe­(II) center, differing from the analogous WT CDO species in the presence of an additional water ligand. The enhanced affinity of the Cys-bound Fe­(II) center for a sixth ligand in the H155A CDO variant likely stems from the increased level of conformational freedom of the cysteine–tyrosine cross-link in the absence of the H155 imidazole ring. Notably, the nitrosyl adduct of Cys-bound Fe­(II)­CDO [which mimics the (O₂/Cys)–CDO intermediate] is essentially unaffected by the H155A substitution, suggesting that the primary role played by the H155 side chain in CDO catalysis is to discourage the binding of a water molecule to the Cys-bound Fe­(II)­CDO active site