O2-dependent Oxidation Research Articles

Improved resolution of 3-mercaptopropionate dioxygenase active site provided by ENDOR spectroscopy offers insight into catalytic mechanism

3-mercaptopropionate (3MPA) dioxygenase (MDO) is a mononuclear nonheme iron enzyme that catalyzes the O2-dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. MDO is a member of the cysteine dioxygenase family of small molecule thiol dioxygenases and thus shares a conserved sequence of active site residues (Serine-155, Histidine-157, and Tyrosine-159), collectively referred to as the SHY-motif. It has been demonstrated that these amino acids directly interact with the mononuclear Fe-site, influencing steady-state catalysis, catalytic efficiency, O2-binding, and substrate coordination. However, the underlying mechanism by which this is accomplished is poorly understood. Here, pulsed electron paramagnetic resonance spectroscopy [1H Mims electron nuclear double resonance spectroscopy] is applied to validate density functional theory computational models for the MDO Fe-site simultaneously coordinated by substrate and nitric oxide (NO), (3MPA/NO)-MDO. The enhanced resolution provided by electron nuclear double resonance spectroscopy allows for direct observation of Fe-bound substrate conformations and H-bond donation from Tyr159 to the Fe-bound NO ligand. Further inclusion of SHY-motif residues within the validated model reveals a distinct channel restricting movement of the Fe-bound NO-ligand. It has been argued that the iron-nitrosyl emulates the structure of potential Fe(III)-superoxide intermediates within the MDO catalytic cycle. While the merit of this assumption remains unconfirmed, the model reported here offers a framework to evaluate oxygen binding at the substrate-bound Fe-site and possible reaction mechanisms. It also underscores the significance of hydrogen bonding interactions within the enzymatic active site.

Non-standard amino acid incorporation into thiol dioxygenases.

Thiol dioxygenases (TDOs) are non‑heme Fe(II)‑dependent enzymes that catalyze the O2-dependent oxidation of thiol substrates to their corresponding sulfinic acids. Six classes of TDOs have thus far been identified and two, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), are found in eukaryotes. All TDOs belong to the cupin superfamily of enzymes, which share a common β‑barrel fold and two cupin motifs: G(X)5HXH(X)3-6E(X)6G and G(X)5-7PXG(X)2H(X)3N. Crystal structures of TDOs revealed that these enzymes contain a relatively rare, neutral 3‑His iron‑binding facial triad. Despite this shared metal-binding site, TDOs vary greatly in their secondary coordination spheres. Site‑directed mutagenesis has been used extensively to explore the impact of changes in secondary sphere residues on substrate specificity and enzymatic efficiency. This chapter summarizes site-directed mutagenesis studies of eukaryotic TDOs, focusing on the tools and practicality of non‑standard amino acid incorporation.

Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site.

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for L-cysteine.

Biocompatible 2D Cu-TCPP Nanosheets Derived from Cu2O Nanocubes as Multifunctional Nanoplatforms for Combined Anticancer Therapy.

Two-dimensional (2D) metal-organic frameworks (MOFs) could serve as multifunctional nanoplatforms to load small-molecule drugs and enzyme-mimicking nanoparticles (NPs) with a high efficiency for combined cancer therapy. Herein, we have prepared novel 2D Cu-tetrakis (4-carboxyphenyl) porphyrin (TCPP) nanosheets with an average thickness of 1.2 ± 0.1 nm using Cu2O nanocubes (50 nm) as a template and solid copper ion supplier. Cu2O nanocubes can be consumed and hybridized with the obtained Cu-TCPP, depending on the molar ratio of Cu2O and TCPP linker. The resultant Cu2O/Cu-TCPP could serve as nanoplatforms for co-loading of Pt and Au NPs to construct multifunctional Cu2O/Cu-TCPP/(Pt-Au) nanomedicines, which showed a superior anticancer effect via multiple therapeutic modes. For instance, Cu(II)-TCPP can produce 1O2 in the presence of acidic H2O2 by the Russell mechanism and the intrinsic Cu(I) ions (derived from the residual Cu2O) could mediate a Fenton-like reaction in tumorous tissues to generate toxic hydroxyl radicals (•OH). Moreover, the loaded Pt NPs with catalase (CAT)-mimic activity could decompose hydrogen peroxide (H2O2) into O2 within the tumor cells, increasing the local O2 concentration, modulating the tumorous hypoxia atmosphere, and promoting the O2-dependent glucose oxidation reaction. Furthermore, Au NPs with glucose oxidase (GOx)-mimic activity could accelerate the consumption of glucose and cut nutrient supply to induce starvation therapy. Consequently, our designed 2D MOF-based therapeutic nanomedicines would be a promising candidate for future smart and combined cancer therapy.

Coral symbionts evolved a functional polycistronic flavodiiron gene.

Photosynthesis in cyanobacteria, green algae, and basal land plants is protected against excess reducing pressure on the photosynthetic chain by flavodiiron proteins (FLV) that dissipate photosynthetic electrons by reducing O2. In these organisms, the genes encoding FLV are always conserved in the form of a pair of two-type isozymes (FLVA and FLVB) that are believed to function in O2 photo-reduction as a heterodimer. While coral symbionts (dinoflagellates of the family Symbiodiniaceae) are the only algae to harbor FLV in photosynthetic red plastid lineage, only one gene is found in transcriptomes and its role and activity remain unknown. Here, we characterized the FLV genes in Symbiodiniaceae and found that its coding region is composed of tandemly repeated FLV sequences. By measuring the O2-dependent electron flow and P700 oxidation, we suggest that this atypical FLV is active in vivo. Based on the amino-acid sequence alignment and the phylogenetic analysis, we conclude that in coral symbionts, the gene pair for FLVA and FLVB have been fused to construct one coding region for a hybrid enzyme, which presumably occurred when or after both genes were inherited from basal green algae to the dinoflagellate. Immunodetection suggested the FLV polypeptide to be cleaved by a post-translational mechanism, adding it to the rare cases of polycistronic genes in eukaryotes. Our results demonstrate that FLV are active in coral symbionts with genomic arrangement that is unique to these species. The implication of these unique features on their symbiotic living environment is discussed.

Outer-Sphere Tyrosine 159 within the 3-Mercaptopropionic Acid Dioxygenase S-H-Y Motif Gates Substrate-Coordination Denticity at the Non-Heme Iron Active Site.

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Regardless of the phylogenic domain, the active site for this enzyme class is typically comprised of two major features: (1) a mononuclear ferrous iron coordinated by three protein-derived histidines and (2) a conserved sequence of outer Fe-coordination-sphere amino acids (Ser-His-Tyr) spatially adjacent to the iron site (∼3 Å). Here, we utilize a promiscuous 3-mercaptopropionic acid dioxygenase cloned from Azotobacter vinelandii (Av MDO) to explore the function of the conserved S-H-Y motif. This enzyme exhibits activity with 3-mercaptopropionic acid (3mpa), l-cysteine (cys), as well as several other thiol-bearing substrates, thus making it an ideal system to study the influence of residues within the highly conserved S-H-Y motif (H157 and Y159) on substrate specificity and reactivity. The pKa values for these residues were determined by pH-dependent steady-state kinetics, and their assignments verified by comparison to H157N and Y159F variants. Complementary electron paramagnetic resonance and Mössbauer studies demonstrate a network of hydrogen bonds connecting H157-Y159 and Fe-bound ligands within the enzymatic Fe site. Crucially, these experiments suggest that the hydroxyl group of Y159 hydrogen bonds to Fe-bound NO and, by extension, Fe-bound oxygen during native catalysis. This interaction alters both the NO binding affinity and rhombicity of the 3mpa-bound iron-nitrosyl site. In addition, Fe coordination of cys is switched from thiolate only to bidentate (thiolate/amine) for the Y159F variant, indicating that perturbations within the S-H-Y proton relay network also influence cys Fe binding denticity.

Bioinspired oxidation of oximes to nitric oxide with dioxygen by a nonheme iron(II) complex.

The ability of two iron(II) complexes, [(TpPh2)FeII(benzilate)] (1) and [(TpPh2)(FeII)2(NPP)3] (2) (TpPh2 = hydrotris(3,5-diphenylpyrazol-1-yl)borate, NPP-H = α-isonitrosopropiophenone), of a monoanionic facial N3 ligand in the O2-dependent oxidation of oximes is reported. The mononuclear complex 1 reacts with dioxygen to decarboxylate the iron-coordinated benzilate. The oximate-bridged dinuclear complex (2), which contains a high-spin (TpPh2)FeII unit and a low-spin iron(II)-oximate unit, activates dioxygen at the high-spin iron(II) center. Both the complexes exhibit the oxidative transformation of oximes to the corresponding carbonyl compounds with the incorporation of one oxygen atom from dioxygen. In the oxidation process, the oxime units are converted to nitric oxide (NO) or nitroxyl (HNO). The iron(II)-benzilate complex (1) reacts with oximes to afford HNO, whereas the iron(II)-oximate complex (2) generates NO. The results described here suggest that the oxidative transformation of oximes to NO/HNO follows different pathways depending upon the nature of co-ligand/reductant.Graphic abstract.

Evidence that a respiratory shield in Escherichia coli protects a low-molecular-mass FeII pool from O2-dependent oxidation

Iron is critical for virtually all organisms, yet major questions remain regarding the systems-level understanding of iron in whole cells. Here, we obtained Mössbauer and EPR spectra of Escherichia coli cells prepared under different nutrient iron concentrations, carbon sources, growth phases, and O2 concentrations to better understand their global iron content. We investigated WT cells and those lacking Fur, FtnA, Bfr, and Dps proteins. The coarse-grain iron content of exponentially growing cells consisted of iron-sulfur clusters, variable amounts of nonheme high-spin FeII species, and an unassigned residual quadrupole doublet. The iron in stationary-phase cells was dominated by magnetically ordered FeIII ions due to oxyhydroxide nanoparticles. Analysis of cytosolic extracts by size-exclusion chromatography detected by an online inductively coupled plasma mass spectrometer revealed a low-molecular-mass (LMM) FeII pool consisting of two iron complexes with masses of ∼500 (major) and ∼1300 (minor) Da. They appeared to be high-spin FeII species with mostly oxygen donor ligands, perhaps a few nitrogen donors, and probably no sulfur donors. Surprisingly, the iron content of E. coli and its reactivity with O2 were remarkably similar to those of mitochondria. In both cases, a "respiratory shield" composed of membrane-bound iron-rich respiratory complexes may protect the LMM FeII pool from reacting with O2 When exponentially growing cells transition to stationary phase, the shield deactivates as metabolic activity declines. Given the universality of oxidative phosphorylation in aerobic biology, the iron content and respiratory shield in other aerobic prokaryotes might be similar to those of E. coli and mitochondria.

62 - Generation of superoxide radical by human cytochrome b5 reductase is modulated by cytochrome b5

Cytochrome b5 reductase (CYB5R) plays a central role in many diverse metabolic reactions by coupling NAD(P)H oxidation to reduction of proteins, such as cytochrome b5 (CYB5). Together, CYB5R and CYB5 transfer electrons to terminal oxidases, such as the mitochondrial amidoxime reducing component (mARC) or hemoglobin. Previous studies have demonstrated that nitric oxide (NO) production by the mARC/CYB5/CYB5R system is quenched by molecular oxygen (O2) and suggested that superoxide radical production was involved, as the addition of Cu,Zn-superoxide(SOD1) dismutase partially recovered NO production. Flavin proteins are known to generate superoxide, suggesting that the CYB5R was the likely site of superoxide radical production. However, the direct formation of superoxide by human CYB5R had not been studied. Here, studies were performed to understand the related mechanisms of radical generation by CYB5R in the absence and presence of CYB5. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with CYB5R and NADH. An EPR spectrum corresponding to the superoxide radical adduct of DMPO (DMPO-OOH) was obtained. This EPR signal was quenched by the addition of the SOD1. The amount of DMPO-OOH formed from the oxidation of NADH by the CYB5R increased with NADH concentration. Importantly, the formation of DMPO-OOH was abrogated in the presence of CYB5. Spectral kinetics demonstrate that NADH consumption is O2-dependent, as no NADH consumption was observed in the absence of O2. From these results, we propose a mechanism in which NADH-reduced CYB5R catalyzes superoxide radical formation. Our results demonstrate that human CYB5R catalyzes the O2-dependent oxidation of NADH to generate superoxide radical. Additionally, CYB5 may protect biological targets from oxidative damage, by inhibiting CYB5R superoxide radical production. The ability of CYB5R to catalyze superoxide radical production in response to O2 has potential pathophysiological implications for the ischemia/reperfusion induced damages, endothelial dysfunction, and cardiovascular and neurological diseases.

Rational Engineering of a Flavoprotein Oxidase for Improved Direct Oxidation of Alcohols to Carboxylic Acids.

The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O2-dependent oxidation of prim-alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to “over-oxidation” of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and Michaelis–Menten kinetics were performed to corroborate these findings.

Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O2-dependent oxidation of primary alcohols

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Previous chemical rescue studies identified a putative FeIII-O2− intermediate that precedes substrate oxidation in Mus musculus cysteine dioxygenase (Mm CDO). Given that a similar reactive intermediate has been identified in the extradiol dioxygenase 2, 3-HCPD, it is conceivable that these enzymes share other mechanistic features with regard to substrate oxidation. To explore this possibility, enzymatic reactions with Mm CDO (as well as the bacterial 3-mercaptopropionic acid dioxygenase, Av MDO) were performed using a substrate analogue (2-mercaptoaniline, 2ma). This aromatic thiol closely approximates the catecholic substrate of homoprotocatechuate of 2, 3-HPCD while maintaining the 2-carbon thiol-amine separation preferred by Mm CDO. Remarkably, both enzymes exhibit 2ma-gated O2-consumption; however, none of the expected products for thiol dioxygenase or intra/extradiol dioxygenase reactions were observed. Instead, benzothiazoles are produced by the condensation of 2ma with aldehydes formed by an off-pathway oxidation of primary alcohols added to aqueous reactions to solubilize the substrate. The observed oxidation of 1º-alcohols in 2ma-reactions is consistent with the formation of a high-valent intermediate similar to what has been reported for cytochrome P450 and mononuclear iron model complexes.

Comparison of Nitrogen Oxide Metabolism among Diverse Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) have well characterized genes that encode and express nitrite reductases (NIR) and nitric oxide reductases (NOR). However, the connection between presence or absence of these and other genes for nitrogen transformations with the physiological production of nitric oxide (NO) and nitrous oxide (N2O) has not been tested across AOB isolated from various trophic states, with diverse phylogeny, and with closed genomes. It is therefore unclear if genomic content for nitrogen oxide metabolism is predictive of net N2O production. Instantaneous microrespirometry experiments were utilized to measure NO and N2O emitted by AOB during active oxidation of ammonia (NH3) or hydroxylamine (NH2OH) and through a period of anoxia. This data was used in concert with genomic content and phylogeny to assess whether taxonomic factors were predictive of nitrogen oxide metabolism. Results showed that two oligotrophic AOB strains lacking annotated NOR-encoding genes released large quantities of NO and produced N2O abiologically at the onset of anoxia following NH3-oxidation. Furthermore, high concentrations of N2O were measured during active O2-dependent NH2OH oxidation by the two oligotrophic AOB in contrast to non-oligotrophic strains that only produced N2O at the onset of anoxia. Therefore, complete nitrifier denitrification did not occur in the two oligotrophic strains, but did occur in meso- and eutrophic strains, even in Nitrosomonas communis Nm2 that lacks an annotated NIR-encoding gene. Regardless of mechanism, all AOB strains produced measureable N2O under tested conditions. This work further confirms that AOB require NOR activity to enzymatically reduce NO to N2O in the nitrifier denitrification pathway, and also that abiotic reactions play an important role in N2O formation, in oligotrophic AOB lacking NOR activity.

Anoxic oxidation of chromium

Abstract Naturally occurring Cr(VI) has been ascribed to terrestrial Cr(III) oxidation by Mn (di)oxides, generated through reaction of Mn(II) with molecular oxygen (O2). However, hydrogen peroxide (H2O2) is a potential oxidant of Cr(III) that may form in serpentinization (high H2, low O2) systems where chromite [i.e., the main mineralogical source of Cr(III)] is abundant. Accordingly, here we evaluate H2O2 and chromite interactions in serpentinization systems to determine pathways of Cr(III) oxidation that alters the current paradigm of O2-dependent oxidation. Field observations support that metastable H2O2 and Cr(VI) are present in serpentinization-related fluids relatively absent of O2. Further, laboratory experiments demonstrate and support that H2O2 is a kinetically facile oxidant of chromite, especially under alkaline conditions, which provides a variety of alternative means by which Cr(VI) may be generated and supplied to the oceans not directly linked to atmospheric O2. Thus, Cr(III) oxidation pathways, and their influence on the Cr isotopic record, must account for anoxic Cr(III) oxidation in serpentinization systems as well as a variety of H2O2-induced Cr(III) oxidation pathways that may occur in both terrestrial and marine systems.

Non-chemical proton-dependent steps prior to O2-activation limit Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase (MDO) catalysis

3-mercaptopropionate dioxygenase from Azotobacter vinelandii (Av MDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of 3-mercaptopropionate (3mpa) to produce 3-sulfinopropionic acid (3spa). With one exception, the active site residues of MDO are identical to bacterial cysteine dioxygenase (CDO). Specifically, the CDO Arg-residue (R50) is replaced by Gln (Q67) in MDO. Despite this minor active site perturbation, substrate-specificity of Av MDO is more relaxed as compared to CDO. In order to investigate the relative timing of chemical and non-chemical events in Av MDO catalysis, the pH/D-dependence of steady-state kinetic parameters (kcat and kcat/KM) and viscosity effects are measured using two different substrates [3mpa and l-cysteine (cys)]. The pL-dependent activity of Av MDO in these reactions can be rationalized assuming a diprotic enzyme model in which three ionic forms of the enzyme are present [cationic, E(z+1); neutral, Ez; and anionic, E(z−1)]. The activities observed for each substrate appear to be dominated by electrostatic interactions within the enzymatic active site. Given the similarity between MDO and the more extensively characterized mammalian CDO, a tentative model for the role of the conserved ‘catalytic triad’ is proposed.

The "Gln-Type" Thiol Dioxygenase from Azotobacter vinelandii is a 3-Mercaptopropionic Acid Dioxygenase.

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine to produce cysteinesulfinic acid. Bacterial CDOs have been subdivided as either "Arg-type" or "Gln-type" on the basis of the identity of conserved active site residues. To date, "Gln-type" enzymes remain largely uncharacterized. It was recently noted that the "Gln-type" enzymes are more homologous with another thiol dioxygenase [3-mercaptopropionate dioxygenase (MDO)] identified in Variovorax paradoxus, suggesting that enzymes of the "Gln-type" subclass are in fact MDOs. In this work, a putative "Gln-type" thiol dioxygenase from Azotobacter vinelandii (Av) was purified to homogeneity and characterized. Steady-state assays were performed using three substrates [3-mercaptopropionic acid (3mpa), l-cysteine (cys), and cysteamine (ca)]. Despite comparable maximal velocities, the "Gln-type" Av enzyme exhibited a specificity for 3mpa (kcat/KM = 72000 M(-1) s(-1)) nearly 2 orders of magnitude greater than those for cys (110 M(-1) s(-1)) and ca (11 M(-1) s(-1)). Supporting X-band electron paramagnetic resonance (EPR) studies were performed using nitric oxide (NO) as a surrogate for O2 binding to confirm obligate-ordered addition of substrate prior to NO. Stoichimetric addition of NO to solutions of 3mpa-bound enzyme quantitatively yields an iron-nitrosyl species (Av ES-NO) with EPR features consistent with a mononuclear (S = (3)/2) {FeNO}(7) site. Conversely, two distinct substrate-bound conformations were observed in Av ES-NO samples prepared with cys and ca, suggesting heterogeneous binding within the enzymatic active site. Analytical EPR simulations are provided to establish the relative binding affinity for each substrate (3map > cys > ca). Both kinetic and spectroscopic results presented here are consistent with 3mpa being the preferred substrate for this enzyme.

Oxidative uncoupling in cysteine dioxygenase is gated by a proton-sensitive intermediate.

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine (Cys) to produce cysteine sulfinic acid (CSA). This enzyme catalyzes the first committed step in Cys catabolism; thus, it is central to mammalian sulfur metabolism and redox homeostasis. Ironically, despite nearly 45 years of continued research on CDO, essentially no information has been reported with respect to its kinetic mechanism. In this work, the timing of chemical steps in the CDO kinetic mechanism is investigated by pH/pD-dependent steady-state kinetics and solvent isotope effects on kcat, kcat/KM, and (O2/CSA) coupling. Normal solvent kinetic isotope effects of 1.45 ± 0.05 and 2.0 ± 0.1 are observed in kcat-pL and kcat/KM-pL profiles, respectively. Proton inventory experiments within the pL-independent region (pL 8.5) suggest multiple solvent-exchangeable protons in flight for both kcat and kcat/KM data. The influence of solvent viscosity was also investigated to probe non-chemical steps and to verify that the apparent isotope effects were not attributable to increased solvent viscosity of D2O reactions relative to H2O. Although solvent viscosity did have a modest influence on kcat and kcat/KM, the response is not sufficient to account for the observed solvent isotope effects. This suggests that product release is only partially rate-limiting for CDO catalysis. Most crucially, proton inventory of (O2/CSA) coupling indicates that a proton-sensitive transition state directly follows O2 activation. Thus, protonation of a transient species preceding Cys oxidation is gated by protons in flight. This behavior provides valuable insight into the kinetically masked transients generated during catalysis.

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 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.

The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase catalyzes the O2-dependent oxidation of l-tryptophan (l-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high l-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with l-tryptophan (l-Trp) can be accounted for by the sequential, ordered binding of O2 and l-Trp. Analysis of the data shows that at low concentrations of l-Trp, O2 binds first followed by the binding of l-Trp; at higher concentrations of l-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (Em0) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between Em0 (that increases in the presence of l-Trp) and the rate constant for O2 binding. This means that the initial formation of ferric superoxide (Fe3+–O2•–) from Fe2+-O2 becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (kcat and Em0) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.

The O 2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O(2), FNR binds a [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O(2), FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S](2+) cluster. The reaction of the [4Fe-4S](2+) cluster with O(2) has been shown to proceed via a 2-step process, an O(2)-dependent 1-electron oxidation to yield a [3Fe-4S](+) intermediate with release of 1 Fe(2+) ion, followed by spontaneous rearrangement to the [2Fe-2S](2+) form with release of 1 Fe(3+) and 2 S(2-) ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [4Fe-4S](2+) cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [4Fe-4S](2+) cluster in the presence of O(2). Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with O(2) is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of O(2) interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo O(2) concentrations.

Reassessment of the Reaction Mechanism in the Heme Dioxygenases

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme enzymes that catalyze the O(2)-dependent oxidation of L-tryptophan to N-formyl-kynurenine. Previous proposals for the mechanism of this reaction have suggested that deprotonation of the indole NH group, either by an active-site base or by oxygen bound to the heme iron, as the initial step. In this work, we have examined the activity of 1-Me-L-Trp with three different heme dioxygenases and their site-directed variants. We find, in contrast to previous work, that 1-Me-L-Trp is a substrate for the heme dioxygenase enzymes. These observations suggest that deprotonation of the indole N(1) is not essential for catalysis, and an alternative reaction mechanism, based on the known chemistry of indoles, is presented.