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.

Influence of cysteine 164 on active site structure in rat cysteine dioxygenase

“…Either way, the C164S variant is unable to deprotonate in this pH range and/or form this disulfide and therefore is able to be fully crosslinked, in contrast to WT. In contrast to our results with WT, Pierce and coworkers have been able to form fully crosslinked WT mouse CDO, [11] something we have been unable to do using our protein and methods.…”

“…Nonetheless, we and others have shown that mammalian CDO activity is dependent upon the amount of crosslink. [11,12] Indeed, the kinetic parameters of the two isoforms were shown to be quite different. [13] A second posttranslational modification forms in mammalian CDO; a disulfide between cysteine 164 and exogenous cysteine.…”

Spectroscopic and Computational Investigation of Iron(III) Cysteine Dioxygenase: Implications for the Nature of the Putative Superoxo-Fe(III) Intermediate

“…This species exhibits a g -spread similar to, though slightly smaller than, that of the (N 3 – /Cys)-Fe(III)CDO adduct (Δ g = 0.442 for the cyanide adduct, as compared to Δ g = 0.547 for the azide-bound species). 18 The small differences in g -spread likely reflect a more delocalized “SOMO” and/or smaller splitting of the “t 2g set” of Fe 3d-based MOs (due to π-backbonding) in the (CN – /Cys)-Fe(III)CDO species.…”

“…19 Although eukaryotic forms of CDO are catalytically competent if the cross-link is not formed, enzymatic activity increases drastically (>20-fold) when the cross-link is present. 18 Notably, prokaryotic versions of CDO lack this cross-link, yet they maintain a similar level of activity as eukaryotic, cross-linked CDO. 20 …”

“…21 This strategy has recently been employed by exposing Cys-Fe(III)CDO to cyanide, which led to the formation of an S = 1 / 2 species whose electronic structure was found to be sensitive to the absence or presence of the C–Y cross-link. 18 Furthermore, an electron paramagnetic resonance (EPR)-based investigation of a superoxide- and Cys-bound Fe(III)CDO complex generated in situ demonstrated that this species is capable of turning over, albeit slowly, strongly suggesting that a (superoxo/Cys)-Fe(III)CDO intermediate is formed in the catalytic cycle. 24 Additional support for the involvement of such an intermediate has been obtained in a recent investigation of the NO adducts of Cys- and Sec-Fe(II)CDO.…”