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

Abstract

Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron-dependent enzyme that converts exogenous cysteine (Cys) to cysteine sulfinic acid using molecular oxygen. Although the complete catalytic mechanism is not yet known, several recent reports presented evidence for an Fe(III)-superoxo reaction intermediate. In this work, we have utilized spectroscopic and computational methods to investigate the as-isolated forms of CDO, as well as Cys-bound Fe(III)CDO, both in the absence and presence of azide (a mimic of superoxide). An analysis of our electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance data of the azide-treated as-isolated forms of CDO within the framework of density functional theory (DFT) computations reveals that azide coordinates directly to the Fe(III), but not the Fe(II) center. An analogous analysis carried out for Cys-Fe(III)CDO provides compelling evidence that at physiological pH, the iron center is six coordinate, with hydroxide occupying the sixth coordination site. Upon incubation of this species with azide, the majority of the active sites retain hydroxide at the iron center. Nonetheless, a modest perturbation of the electronic structure of the Fe(III) center is observed, indicating that azide ions bind near the active site. Additionally, for a small fraction of active sites, azide displaces hydroxide and coordinates directly to the Cys-bound Fe(III) center to generate a low-spin (S = 1/2) Fe(III) complex. In the DFT-optimized structure of this complex, the central nitrogen atom of the azide moiety lies within 3.12 Å of the cysteine sulfur. A similar orientation of the superoxide ligand in the putative Fe(III)-superoxo reaction intermediate would promote the attack of the distal oxygen atom on the sulfur of substrate Cys.

Structure-Based Insights into the Role of the Cys–Tyr Crosslink and Inhibitor Recognition by Mammalian Cysteine Dioxygenase

“…Soaks of C93A, Y157F and wild-type CDO at pH 8.0 with homocysteine (Hcy), a known competitive inhibitor, 19; 39; 49 were solved at resolutions between 1.3 and 1.65 Å (Table 2). All of the complexes showed a very similar binding mode with only the thiol coordinating the iron (~2.20 Å) and the α-amino group being ~3.5 Å from the iron (Figure 6A-C).…”

“…40 Crystal soaks with 100 mM azide at pH 6.2 showed no binding to the C93A or Y157F variants, but yielded a 1.5 Å resolution full occupancy azide-bound structure of wild-type CDO (Figure 6E and Table 2). In the azide-bound structure, a terminal nitrogen coordinates the iron and the azide extends at a 105° angle from the iron, similar to what has been seen in 3-His 1-carboxylate mono-iron enzymes (e.g.…”

“…We also provide insight into the specificity of the enzyme through determining structures of wild-type CDO and/or the variants in the presence of the competitive inhibitors homocysteine, azide, thiosulfate and D-Cys. 19; 39; 40 …”

Spectroscopic and Electronic Structure Study of ETHE1: Elucidating the Factors Influencing Sulfur Oxidation and Oxygenation in Mononuclear Nonheme Iron Enzymes

“…Heterolytic O-O bond cleavage follows to form a ferryl oxo and sulfoxide. 25–28 Recently, a short-lived intermediate was detected in the reaction of CDO/Cys with O 2 with absorption maxima at 500 and 640 nm. The intermediate could not be further characterized, but time-dependent density functional theory (TD-DFT) calculations predict it to be the peroxo-bridged intermediate that precedes the ferryl oxo/sulfoxide.…”

“…The calculated reaction coordinate showed similarities to those defined for CDO, which utilizes a 3-His triad and bidentate bound substrate. 25–28 However, the ETHE1 active site structure defined here is similar to that of IPNS, which catalyzes a sulfur oxidation on a monodentate bound substrate, utilizing a 2-His/1-Asp facial triad. This led us to perform a series of calculations that identified how these sulfur transforming enzymes select for sulfur oxidation vs. oxygenation.…”