In Situ Structural Observation of a Substrate- and Peroxide-Bound High-Spin Ferric-Hydroperoxo Intermediate in the P450 Enzyme CYP121.

The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon-carbon (C-C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme-substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C-C bond coupling chemistry.

Kinetic and Spectroscopic Characterization of the Catalytic Ternary Complex of Tryptophan 2,3-Dioxygenase.

The first step of the kynurenine pathway for l-tryptophan (l-Trp) degradation is catalyzed by heme-dependent dioxygenases, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase. In this work, we employed stopped-flow optical absorption spectroscopy to study the kinetic behavior of the Michaelis complex of Cupriavidus metallidurans TDO (cmTDO) to improve our understanding of oxygen activation and initial oxidation of l-Trp. On the basis of the stopped-flow results, rapid freeze-quench (RFQ) experiments were performed to capture and characterize this intermediate by Mössbauer spectroscopy. By incorporating the chlorite dismutase-chlorite system to produce high concentrations of solubilized O2, we were able to capture the Michaelis complex of cmTDO in a nearly quantitative yield. The RFQ-Mössbauer results confirmed the identity of the Michaelis complex as an O2-bound ferrous species. They revealed remarkable similarities between the electronic properties of the Michaelis complex and those of the O2 adduct of myoglobin. We also found that the decay of this reactive intermediate is the rate-limiting step of the catalytic reaction. An inverse α-secondary substrate kinetic isotope effect was observed with a kH/kD of 0.87 ± 0.03 when (indole-d5)-l-Trp was employed as the substrate. This work provides an important piece of spectroscopic evidence of the chemical identity of the Michaelis complex of bacterial TDO.

Biocatalytic Carbon-Hydrogen and Carbon-Fluorine Bond Cleavage through Hydroxylation Promoted by a Histidyl-Ligated Heme Enzyme.

LmbB2 is a peroxygenase-like enzyme that hydroxylates L-tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) in the presence of hydrogen peroxide. However, its heme cofactor is ligated by a proximal histidine, not cysteine. We show that LmbB2 can oxidize L-tyrosine analogs with ring-deactivated substituents such as 3-nitro-, fluoro-, chloro-, iodo-L-tyrosine. We also found that the 4-hydroxyl group of the substrate is essential for reacting with the heme-based oxidant and activating the aromatic C-H bond. The most interesting observation of this study was obtained with 3-fluoro-L-tyrosine as a substrate and mechanistic probe. The LmbB2-mediated catalytic reaction yielded two hydroxylated products with comparable populations, i.e., oxidative C-H bond cleavage at C5 to generate 3-fluoro-5-hydroxyl-L-tyrosine and oxygenation at C3 concomitant with a carbon-fluorine bond cleavage to yield DOPA and fluoride. An iron protein-mediated hydroxylation on both C-H and C-F bonds with multiple turnovers is unprecedented. Thus, this finding reveals a significant potential of biocatalysis in C-H/C-X bond (X = halogen) cleavage. Further 18O-labeling results suggest that the source of oxygen for hydroxylation is a peroxide, and that a commonly expected oxidation by a high-valent iron intermediate followed by hydrolysis is not supported for the C-F bond cleavage. Instead, the C-F bond cleavage is proposed to be initiated by a nucleophilic aromatic substitution mediated by the iron-hydroperoxo species. Based on the experimental results, two mechanisms are proposed to explain how LmbB2 hydroxylates the substrate and cleaves C-H/C-F bond. This study broadens the understanding of heme enzyme catalysis and sheds light on enzymatic applications in medicinal and environmental fields.

Probing Ligand Exchange in the P450 Enzyme CYP121 from Mycobacterium tuberculosis: Dynamic Equilibrium of the Distal Heme Ligand as a Function of pH and Temperature.

CYP121 is a cytochrome P450 enzyme from Mycobacterium tuberculosis that catalyzes the formation of a C-C bond between the aromatic groups of its cyclodityrosine substrate (cYY). The crystal structure of CYP121 in complex with cYY reveals that the solvent-derived ligand remains bound to the ferric ion in the enzyme-substrate complex. Whereas in the generally accepted P450 mechanism, binding of the primary substrate in the active-site triggers the release of the solvent-derived ligand, priming the metal center for reduction and subsequent O2 binding. Here we employed sodium cyanide to probe the metal-ligand exchange of the enzyme and the enzyme-substrate complex. The cyano adducts were characterized by UV-vis, EPR, and ENDOR spectroscopies and X-ray crystallography. A 100-fold increase in the affinity of cyanide binding to the enzyme-substrate complex over the ligand-free enzyme was observed. The crystal structure of the [CYP121(cYY)CN] ternary complex showed a rearrangement of the substrate in the active-site, when compared to the structure of the binary [CYP121(cYY)] complex. Transient kinetic studies showed that cYY binding resulted in a lower second-order rate constant (kon (CN)) but a much more stable cyanide adduct with 3 orders of magnitude slower koff (CN) rate. A dynamic equilibrium between multiple high- and low-spin species for both the enzyme and enzyme-substrate complex was also observed, which is sensitive to changes in both pH and temperature. Our data reveal the chemical and physical properties of the solvent-derived ligand of the enzyme, which will help to understand the initial steps of the catalytic mechanism.

Cross-linking of dicyclotyrosine by the cytochrome P450 enzyme CYP121 from Mycobacterium tuberculosis proceeds through a catalytic shunt pathway.

CYP121, the cytochrome P450 enzyme in Mycobacterium tuberculosis that catalyzes a single intramolecular C-C cross-linking reaction in the biosynthesis of mycocyclosin, is crucial for the viability of this pathogen. This C-C coupling reaction represents an expansion of the activities carried out by P450 enzymes distinct from oxygen insertion. Although the traditional mechanism for P450 enzymes has been well studied, it is unclear whether CYP121 follows the general P450 mechanism or uses a different catalytic strategy for generating an iron-bound oxidant. To gain mechanistic insight into the CYP121-catalyzed reaction, we tested the peroxide shunt pathway by using rapid kinetic techniques to monitor the enzyme activity with its substrate dicyclotyrosine (cYY) and observed the formation of the cross-linked product mycocyclosin by LC-MS. In stopped-flow experiments, we observed that cYY binding to CYP121 proceeds in a two-step process, and EPR spectroscopy indicates that the binding induces active site reorganization and uniformity. Using rapid freeze-quenching EPR, we observed the formation of a high-spin intermediate upon the addition of peracetic acid to the enzyme-substrate complex. This intermediate exhibits a high-spin (S = 5/2) signal with g values of 2.00, 5.77, and 6.87. Likewise, iodosylbenzene could also produce mycocyclosin, implicating compound I as the initial oxidizing species. Moreover, we also demonstrated that CYP121 performs a standard peroxidase type of reaction by observing substrate-based radicals. On the basis of these results, we propose plausible free radical-based mechanisms for the C-C bond coupling reaction.

Hypertryptophanemia due to tryptophan 2,3-dioxygenase deficiency.

In this report we describe the first human case of hypertryptophanemia confirmed to be due to tryptophan 2,3-dioxygenase deficiency. The underlying etiology was established by sequencing the TDO2 gene, in which there was compound heterozygosity for two rare variants: c.324G>C, p.Met108Ile and c.491dup, p.Ile165Aspfs*12. The pathogenicity of these variants was confirmed by molecular-level studies, which showed that c.491dup does not produce soluble protein and c.324G>C results in a catalytically less efficient Met108Ile enzyme that is prone to proteolytic degradation. The biochemical phenotype of hypertryptophanemia and hyperserotoninemia does not appear to have significant clinical consequences.

An unexpected copper catalyzed 'reduction' of an arylazide to amine through the formation of a nitrene intermediate.

Azido nitrobenzoxadiazole (NBD) was observed to undergo a 'reduction' reaction in the absence of an obvious reducing agent, leading to amine formation. In the presence of an excess amount of DMSO, a sulfoxide conjugate was also formed. The ratio of these two products was both temperature- and solvent-dependent, with the addition of water significantly enhancing the ratio of the 'reduction' product. Two intermediates of the azido-NBD reaction in DMSO were trapped and characterized by low-temperature EPR spectroscopy. One was an organic free radical (S=1/2) and another was a triplet nitrene (S=1) species. A mechanism was proposed based on the characterized free radical and triplet intermediates.

Tryptophan-mediated charge-resonance stabilization in the bis-Fe(IV) redox state of MauG.

The diheme enzyme MauG catalyzes posttranslational modifications of a methylamine dehydrogenase precursor protein to generate a tryptophan tryptophylquinone cofactor. The MauG-catalyzed reaction proceeds via a bis-Fe(IV) intermediate in which one heme is present as Fe(IV)=O and the other as Fe(IV) with axial histidine and tyrosine ligation. Herein, a unique near-infrared absorption feature exhibited specifically in bis-Fe(IV) MauG is described, and evidence is presented that it results from a charge-resonance-transition phenomenon. As the two hemes are physically separated by 14.5 Å, a hole-hopping mechanism is proposed in which a tryptophan residue located between the hemes is reversibly oxidized and reduced to increase the effective electronic coupling element and enhance the rate of reversible electron transfer between the hemes in bis-Fe(IV) MauG. Analysis of the MauG structure reveals that electron transfer via this mechanism is rapid enough to enable a charge-resonance stabilization of the bis-Fe(IV) state without direct contact between the hemes. The finding of the charge-resonance-transition phenomenon explains why the bis-Fe(IV) intermediate is stabilized in MauG and does not permanently oxidize its own aromatic residues.

Chemical rescue of the distal histidine mutants of tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent enzyme that catalyzes the oxidative degradation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK). A highly conserved histidine residue in the distal heme pocket has attracted great attention in the mechanistic studies of TDO. However, a consensus has not been reached regarding whether and how this distal histidine plays a catalytic role after substrate binding. In this study, three mutant proteins, H72S, H72N, and Q73F were generated to investigate the function of the distal histidine residue in Cupriavidus metallidurans TDO (cmTDO). Spectroscopic characterizations, enzymatic kinetic analysis, and chemical rescue assays were employed to study the biochemical properties of the wild-type enzyme and the mutant proteins. Rapid kinetic methods were utilized to explore the molecular basis for the observed stimulation of catalytic activity by 2-methylimidazole in the His72 variants. The results indicate that the distal histidine plays multiple roles in cmTDO. First, His72 contributes to but is not essential for substrate binding. In addition, it shields the heme center from nonproductive binding of exogenous small ligand molecules (i.e., imidazole and its analogs) via steric hindrance. Most importantly, His72 participates in the subsequent chemical catalytic steps after substrate binding possibly by providing H-bonding interactions to the heme-bound oxygen.

Enzyme reactivation by hydrogen peroxide in heme-based tryptophan dioxygenase.

An intriguing mystery about tryptophan 2,3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2,3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔE(Q) = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. In the presence of L-Trp, the reactivation was demonstrated by enzyme assays and by various spectroscopic techniques. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism. These results may shed light on how a metalloenzyme maintains its catalytic activity in an oxidizing environment.