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

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 (<i>S</i> = 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

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

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

Cysteine dioxygenase (CDO) is a mononuclear nonheme iron­(II)-dependent enzyme critical for maintaining appropriate cysteine (Cys) and taurine levels in eukaryotic systems. Because CDO possesses both an unusual 3-His facial ligation sphere to the iron center and a rare Cys–Tyr cross-link 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 <i>S</i> = 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 semiempirical 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 is not oxidized by CDO

Second-Sphere Interactions between the C93–Y157 Cross-Link and the Substrate-Bound Fe Site Influence the O₂ Coupling Efficiency in Mouse Cysteine Dioxygenase

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 <i>k</i>_cat 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 (<i>S</i> = 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 <b>g</b>-tensor relative to the CDO Fe site molecular axis

Time-Resolved Investigations of Heterobimetallic Cofactor Assembly in R2lox Reveal Distinct Mn/Fe Intermediates

The assembly mechanism of the Mn/Fe ligand-binding oxidases (R2lox), a family of proteins that are homologous to the nonheme diiron carboxylate enzymes, has been investigated using time-resolved techniques. Multiple heterobimetallic intermediates that exhibit unique spectral features, including visible absorption bands and exceptionally broad electron paramagnetic resonance signatures, are observed through optical and magnetic resonance spectroscopies. On the basis of comparison to known diiron species and model compounds, the spectra have been attributed to (μ-peroxo)-Mn^III/Fe^III and high-valent Mn/Fe species. Global spectral analysis coupled with isotopic substitution and kinetic modeling reveals elementary rate constants for the assembly of Mn/Fe R2lox under aerobic conditions. A complete reaction mechanism for cofactor maturation that is consistent with experimental data has been developed. These results suggest that the Mn/Fe cofactor can perform direct C–H bond abstraction, demonstrating the potential for potent chemical reactivity that remains unexplored

Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from <i>Streptomyces fradiae</i>, a Cobalamin-Dependent Radical <i>S</i>‑Adenosylmethionine Methylase

Fom3, a cobalamin-dependent radical <i>S</i>-adenosylmethionine (SAM) methylase, has recently been shown to catalyze the methylation of carbon 2″ of cytidylyl-2-hydroxyethylphosphonate (HEP-CMP) to form cytidylyl-2-hydroxypropylphosphonate (HPP-CMP) during the biosynthesis of fosfomycin, a broad-spectrum antibiotic. It has been hypothesized that a 5′-deoxyadenosyl 5′-radical (5′-dA^•) generated from the reductive cleavage of SAM abstracts a hydrogen atom from HEP-CMP to prime the substrate for addition of a methyl group from methylcobalamin (MeCbl); however, the mechanistic details of this reaction remain elusive. Moreover, it has been reported that Fom3 catalyzes the methylation of HEP-CMP to give a mixture of the (<i>S</i>)-HPP and (<i>R</i>)-HPP stereoisomers, which is rare for an enzyme-catalyzed reaction. Herein, we describe a detailed biochemical investigation of a Fom3 that is purified with 1 equiv of its cobalamin cofactor bound, which is almost exclusively in the form of MeCbl. Electron paramagnetic resonance and Mössbauer spectroscopies confirm that Fom3 contains one [4Fe-4S] cluster. Using deuterated enantiomers of HEP-CMP, we demonstrate that the 5′-dA^• generated by Fom3 abstracts the C2″-<i>pro-R</i> hydrogen of HEP-CMP and that methyl addition takes place with inversion of configuration to yield solely (<i>S</i>)-HPP-CMP. Fom3 also sluggishly converts cytidylyl-ethylphosphonate to the corresponding methylated product but more readily acts on cytidylyl-2-fluoroethylphosphonate, which exhibits a lower C2″ homolytic bond-dissociation energy. Our studies suggest a mechanism in which the substrate C2″ radical, generated upon hydrogen atom abstraction by the 5′-dA^•, directly attacks MeCbl to transfer a methyl radical (CH₃^•) rather than a methyl cation (CH₃⁺), directly forming cob­(II)­alamin in the process

Substrate-Triggered Formation of a Peroxo-Fe2(III/III) Intermediate during Fatty Acid Decarboxylation by UndA

The iron-dependent oxidase UndA cleaves one C3-H bond and the C1-C2 bond of dodecanoic acid to produce 1-undecene and CO2. A published X-ray crystal structure showed that UndA has a heme-oxygenase-like fold, thus associating it with a structural superfamily that includes known and postulated non-heme diiron proteins, but revealed only a single iron ion in the active site. Mechanisms proposed for initiation of decarboxylation by cleavage of the C3-H bond using a monoiron cofactor to activate O2 necessarily invoked unusual or potentially unfeasible steps. Here we present spectroscopic, crystallographic, and biochemical evidence that the cofactor of Pseudomonas fluorescens Pf-5 UndA is actually a diiron cluster and show that binding of the substrate triggers rapid addition of O2 to the Fe2(II/II) cofactor to produce a transient peroxo-Fe2(III/III) intermediate. The observations of a diiron cofactor and substrate-triggered formation of a peroxo-Fe2(III/III) intermediate suggest a small set of possible mechanisms for O2, C3-H and C1-C2 activation by UndA; these routes obviate the problematic steps of the earlier hypotheses that invoked a single iron

Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical

All cells obtain 2′-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, β; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in β. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the β is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.National Institutes of Health (Grant GM119707