Spectroscopic and Computational Characterization of the NO Adduct of Substrate-Bound Fe(II) Cysteine Dioxygenase: Insights into the Mechanism of O₂ Activation

Abstract

Cysteine dioxygenase (CDO) is a mononuclear non-heme iron(II)-dependent enzyme critical for maintaining appropriate cysteine (Cys) and taurine levels in eukaryotic systems. Since CDO possesses both an unusual 3-His facial ligation sphere to the iron center and a rare Cys-Tyr crosslink near the active site, the mechanism by which it converts Cys and molecular oxygen to cysteine sulfinic acid is of broad interest. However, as of yet direct experimental support for any of the proposed mechanisms is still lacking. In this study, we have used NO as a substrate analogue for O2 to prepare a species that mimics the geometric and electronic structures of an early reaction intermediate. The resultant unusual S=1/2 {FeNO}7 species was characterized by magnetic circular dichroism, electron paramagnetic resonance, and electronic absorption spectroscopies, as well as computational methods including density functional theory and semi-empirical calculations. The NO adducts of Cys- and selenocysteine (Sec)-bound Fe(II)CDO exhibit virtually identical electronic properties; yet, CDO is unable to oxidize Sec. To explore the differences in reactivity between Cys- and Sec-bound CDO, the geometries and energies of viable O2-bound intermediates were evaluated computationally, and it was found that a low-energy quintet-spin intermediate on the Cys reaction pathway adopts a different geometry for the Sec-bound adduct. The absence of a low-energy O2 adduct for Sec-bound CDO is consistent with our experimental data and may explain why Sec does not act as a substrate for CDO.

Mono- and binuclear non-heme iron chemistry from a theoretical perspective

“…[114,115] O2 activation [116,117] and the preceding required water dissociation [118] was also investigated in the tetrahydrobiopterin-iron amino acid hydroxylases.…”

“…Figure 10: Proposed mechanism of CDO. [114] It was earlier predicted by the theoretical calculations [142,201] that S = 2 Fe IV =O species are more reactive in the oxidative reactions than their S = 1 analogues. It should be, however, noted that the direct experimental evidence is still lacking.…”

“…[168] The inability to bind O2 was found to be the reason why CDO does not convert selenocysteine into the corresponding seleninic acid. [114] In dimanganese class Ib ribonucleotide reductase, the rate by which the H2O2 oxidant is supplied seems to be of key importance in the selectivity toward the native tyrosyl radical generation instead of the possible catalase activity. [239] …”

Mechanism-based inhibition of human persulfide dioxygenase by γ-glutamyl-homocysteinyl-glycine

“…Like PDO, the reaction mechanism of isopencillin N-synthase involves Fe-S-peptide interactions as the linear cysteinyl-tripeptide substrate is transformed into isopenicillin N via desaturative ring closure and concomitant reduction of O 2 to water (22). Fe-SCys interactions are also important in the cysteine dioxygenase-catalyzed dioxygenation of cysteine to cysteinesulfinic acid (23). Both isopencillin Nsynthase and cysteine dioxygenase involve Fe-SCH 2 R complexes versus an Fe-S-SCH 2 R complex in PDO.…”

Reactive Mass Transfer of Single NO Bubbles and Bubble Bouncing in Aqueous Ferric Solutions – A Feasibility Study

“…NO can be regarded as an important cellular signaling molecule involved in many physiological and pathological processes. The interaction of NO with iron centers of hemoproteins [24] and nonheme enzymes with cysteine environment [25] plays an important role in its biological activity. To put it simply, the formation of a Fe(L)-NO bond (L means ligand) triggers the opening of a blood vessel.…”