In-Situ Generation of Hydrogen Polysulfides (H2Sn) with Thioglucose, Glucose Oxidase, and Chloroperoxidase.

Hydrogen polysulfides (H2Sn) have emerged as critical physiological mediators that are closely associated with hydrogen sulfide (H2S) signaling. H2Sn exhibit greater nucleophilicity than H2S while also having electrophilic characteristics, enabling unique activities such as protein S-persulfidation. Despite their physiological importance, mechanisms and reactivities of H2Sn remain inadequately explored due to their inherent instability in aqueous environments. Consequently, there is a need to develop biocompatible methods for controlled H2Sn generation to elucidate their behaviors in biological contexts. Herein, we present a dual enzyme system (containing glucose oxidase (GOx) and chloroperoxidase (CPO)) with thioglucose as the substrate to facilitate the controlled release of H2Sn. Fluorescence measurements with SSP4 and the trapping studies allowed us to confirm the production of H2Sn. Such a method may be useful in elucidating the reactivity of hydrogen polysulfides in biological systems as well as provide a potential delivery of H2Sn to target sites for biological applications.

Chemoenzymatic Synthesis of Sterically Hindered Biaryls by Suzuki Coupling and Vanadium Chloroperoxidase Catalyzed Halogenations.

Halogenated biaryls are vital structural skeletons in bioactive products. In this study, an effective chemoenzymatic halogenation by vanadium-dependent chloroperoxidase from Camponotus inaequalis (CiVCPO) enabled the transformation of freely rotating biaryl bonds to sterically hindered axis. The yields were up to 84 % for the tribrominated biaryl products and up to 65 % when isolated. Furthermore, a one-pot, two-step chemoenzymatic strategy by incorporating transition metal catalyzed Suzuki coupling and the chemoenzymatic halogenation in aqueous phase were described. This strategy demonstrates a simplified one-pot reaction sequence with organometallic and biocatalytic procedures under economical and environmentally beneficial conditions that may inspire further research on synthesis of sterically hindered biaryls.

The chloroperoxidase immobilized on porous carbon nanobowls for the detection of trichloroacetic acid by electroenzymatic synergistic catalysis.

Trichloroacetic acid (TCA), as a by-product of chlorination disinfection, is a highly carcinogenic chemical. Due to the widespread use of chlorination disinfection, it is critical to detect TCA in drinking water to decrease the incidence of disease. In this work, we developed an efficient TCA biosensor via electroenzymatic synergistic catalysis. The porous carbon nanobowls (PCNB) are prepared and wrapped by an amyloid like proteins formed by phase-transitioned lysozyme (PTL-PCNB), then, chloroperoxidase (CPO) is abounding to PTL-PCNB owing to its strong adhesion. The ionic liquid of 1-ethyl-3-methylimidazolium bromide (ILEMB) is co-immobilized on PTL-PCNB to from CPO-ILEMB@PTL-PCNB nanocomposite to assist the direct electron transfer (DET) of CPO. The PCNB plays two roles here. In addition, to increasing the conductivity, it serves as an ideal support for holding CPO; The CPO-ILEMB@PTL-PCNB nanocomposite modified electrode presents high efficiency for sensing TCA. Through electroenzymatic synergistic catalysis, a wide detection range of 33 µmol L-1 to 98 mmol L-1 can be achieved with a low detection limit of 5.9 µmol L-1, and high stability, selectivity as well as reproducibility, which ensures its potential practical applicability. This work provides a new platform for the electro-enzyme synergistic catalysis in one pot.

17O Electron Nuclear Double Resonance Analysis of Compound I: Inverse Correlation between Oxygen Spin Population and Electron Donation.

Although the activation of inert C-H bonds by metal-oxo complexes has been widely studied, important questions remain, particularly regarding the role of oxygen spin population (i.e., unpaired electrons on the oxo ligand) in facilitating C-H bond cleavage. In order to shed light on this issue, we have utilized 17O electron nuclear double resonance spectroscopy to measure the oxygen spin populations of three compound I intermediates in heme enzymes with different reactivities toward C-H bonds: chloroperoxidase, cytochrome P450, and a selenolate (selenocysteinyl)-ligated cytochrome P450. The experimental data suggest an inverse correlation between oxygen spin population and electron donation from the axial ligand. We have explored the implications of this result using a Hückel-type molecular orbital model and constrained density functional theory calculations. These investigations have allowed us to examine the relationship between oxygen spin population, oxygen charge, electron donation from the axial ligand, and reactivity.

A Photochemoenzymatic Hunsdiecker-Borodin-Type Halodecarboxylation of Ferulic Acid.

A photochemoenzymatic halodecarboxylation of ferulic acid was achieved using vanadate-dependent chloroperoxidase as (bio)catalyst and oxygen and organic solvent as sole stoichiometric reagents in a biphasic system. Performance and selectivity were improved through a phase transfer catalyst, reaching a turnover number of 660.000 for the enzyme.

Toward Environmentally Benign Electrophilic Chlorinations: From Chloroperoxidase to Bioinspired Isoporphyrins.

Recent desires to develop environmentally benign procedures for electrophilic chlorinations have encouraged researchers to take inspiration from nature. In particular, the enzyme chloroperoxidase (CPO), which is capable of electrophilic chlorinations through the umpolung of chloride by oxidation with hydrogen peroxide (H2O2), has received lots of attention. CPO itself is unsuitable for industrial use because of its tendency to decompose in the presence of excess H2O2. Biomimetic complexes (CPO active-site mimics) were then developed and have been shown to successfully catalyze electrophilic chlorinations but are too synthetically demanding to be economically viable. Reported efforts at generating the putative active chlorinating agent of CPO (an iron hypochlorite species) via the umpolung of chloride and using simple meso-substituted iron porphyrins were unsuccessful. Instead, a meso-chloroisoporphyrin intermediate was formed, which was shown to be equally capable of performing electrophilic chlorinations. The current developments toward a potential method involving this novel intermediate for environmentally benign electrophilic chlorinations are discussed. Although this novel pathway no longer follows the mechanism of CPO, it was developed from efforts to replicate its function, showing the power that drawing inspiration from nature can have.

Chloroperoxidase-catalyzed oxidative degradation of sulfur mustard.

As a natural heme protein catalyzing the oxidation of sulfides to sulfoxides without sulfone formation, chloroperoxidase (CPO) is well suited for the degradation of sulfur mustard (HD), a persistent chemical warfare agent that has been widely disposed since World War II and continuously leaks into aquatic environments. Herein, we report the first systematic investigation of CPO-catalyzed degradation of HD and the potential application of CPO in destroying chemical weapons under mild conditions. The related Michaelis-Menten parameters (Km=0.17 mM, Vmax=0.06 mM s-1 (R2 =0.935), and kcat= 2717 s-1) indicated nearly a prominent enzymatic efficiency. Under optimal conditions, 80% of HD was transformed to bis(2-chloroethyl) sulfoxide as identified by mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Other metabolites were also generated during the decontamination process. A plausible oxidation mechanism was proposed based on the degradation products, NMR titration experiments, and molecular dynamics simulations. CPO also promoted the degradation of other chemical weapon agents, namely, Lewisite (L) and venomous agent X (VX), thereby exhibiting a broad substrate scope. The high potential of the developed system for the decontamination of aquatic environments was demonstrated by the successful hatching of zebrafish embryos after HD degradation and the survival of zebrafish (Danio rerio, AB strain) larvae after the degradation of Agent Yellow (L+HD).

Amino modified magnetic halloysite nanotube supporting chloroperoxidase immobilization: enhanced stability, reusability, and efficient degradation of pesticide residue in wastewater.

Halloysite nanotube (HNT) is a natural bio-compatible and stable nanomaterial available in abundance at low-cost. In this work, HNT was modified by two strategies to make it suitable for supporting immobilization of chloroperoxidase (CPO). Firstly, Fe3O4 nanoparticles were deposited on HNT, so magnetic separation can be used instead of centrifugation. Then, the magnetic HNT was modified by 3-aminopropyltriethoxysilane (APTES), which can provide amine group on surface of HNT and meanwhile inhibit the agglomeration of magnetic HNT. Then, HNT-Fe3O4 -APTES was linked with branched polyethyleneimine (PEI) to provide more amino for binding with enzyme. The so-prepared CPO@HNT-Fe3O4-APTES-PEI showed enhanced enzyme loading, reusability, improved thermal stability and tolerance to organic solvents than free CPO. For example, after 10 repeated uses, CPO@HNT- Fe3O4-APTES-PEI can maintain 92.20% of its original activity compared with 65.12% of activity of CPO@HNT-APTES-PEI and 45.69% of activity of CPO@HNT. The kinetic parameters indicated the affinity and specificity of immobilized enzyme to substrate was increased. CPO@HNT-Fe3O4-APTES-PEI was very efficient when it was applied in the degradation of pesticides mesotrione in wastewater. The degradation efficiency can reach 90% within 20 min at range of 5-40 µmol·L-1. These results ensure the potential practical application of this bio-materials in wastewater treatment.

A Neutrophil-Inspired Supramolecular Nanogel for Magnetocaloric-Enzymatic Tandem Therapy.

Neutrophils can responsively release reactive oxygen species (ROS) to actively combat infections by exogenous stimulus and cascade enzyme catalyzed bio-oxidation. A supramolecular nanogel is now used as an artificial neutrophil by enzymatic interfacial self-assembly of peptides (Fmoc-Tyr(H2 PO3 )-OH) with magnetic nanoparticles (MNPs) and electrostatic loading of chloroperoxidase (CPO). The MNPs within the nanogel can elevate H2 O2 levels in cancer cells under programmed alternating magnetic field (AMF) similar to the neutrophil activator, and the loaded CPO within protective peptides nanolayer converts the H2 O2 into singlet oxygen (1 O2 ) in a sustained manner for neutrophil-inspired tumor therapy. As a proof of concept study, both the H2 O2 and 1 O2 in cancer cells increase stepwise under a programmed alternating magnetic field. An active enzyme dynamic therapy by magnetically stimulated oxygen stress and sustained enzyme bio-oxidation is thus shown with studies on both cells and animals.

Multiple catalytic roles of chloroperoxidase in the transformation of phenol: Products and pathways.

Chloroperoxidase (CPO) is a hybrid of two different families of enzymes, peroxidases and P450s. However, it is poorly understood on CPO's multiple catalytic functions. Herein, phenol was selected as a model substrate to investigate the multiple catalytic roles of CPO. Results showed that phenol was readily transformed into a variety of brominated organic compounds (BOCs) via the CPO-mediated oxidative process. A total of 16 BOCs were identified using gas and liquid chromatography coupled with mass spectrometry. Possible reaction pathways could be attributable to four CPO-mediated processes, including bromination, radical coupling, intramolecular cyclization and debromination. Higher bromide concentrations and lower pH conditions both facilitated the formation of brominated products. While a higher bromination capacity was observed in pH 3.0 solutions, CPO-mediated radical couplings were more favorable at pH 5.0 and 6.0. Although CPO might catalyze chlorination when chloride and bromide coexisted in the solution, BOCs were the dominant products of CPO-mediated phenol oxidation. Results of this study suggest that various catalytic roles of CPO may contribute to the biotic formation of BOCs in the natural environment.

Positional orientating co-immobilization of bienzyme CPO/GOx on mesoporous TiO2 thin film for efficient cascade reaction.

A multitude of industrial processes are catalyzed by two or more enzymes working together in a cascade way. However, designing efficient enzymatic cascade reactions is still a challenge. In this work, a TiO2 thin film with mesoporous pores was prepared and used as carrier for co-immobilization of chloroperoxidase (CPO) and glucose peroxidase (GOx). By adjusting the dosage of hexadecyltrimethylammonium bromide (CTAB) and the ratio of the two enzymes, CPO and GOx were well distributed and positional orientated to their own appropriate pores to form an ordered "occupation" based on a "feet in right shoes" effect. Moreover, when the pore size was controlled around 12 nm, the enzymes aggregation was inhibited so as to avoid the decrease of activity of enzyme; The catalytic performance of TiO2-GOx and CPO composites was evaluated by the application of decolorization of Orange G dye in a cascaded manner. The oxidant H2O2 needed by CPO is generated in situ through glucose oxidation by GOx. Upon co-immobilization of CPO and GOx on the same carrier, a large increase in the initial catalytic efficiency was detected when compared to an equimolar mixture of the free enzymes, which was four times greater. Moreover, the affinity of the enzyme toward substrate binding was improved according to the kinetic assay. The thermal stability of TiO2-GOx and CPO composites were greatly improved than free enzymes. The TiO2-GOx and CPO composites can be easily separated from the reaction media which facilitate its recycle use.

Partial secretome analysis of Caldariomyces fumago reveals extracellular production of the CPO co-substrate H2O2 and provides a coproduction concept for CPO and glucose oxidase.

The culture supernatant of Caldariomyces fumago strains grown in a minimal medium with fructose contains mainly the biotechnologically relevant enzyme chloroperoxidase (CPO) and only minor amounts of other proteins. Our approach to identify the nature of these proteins via peptide mass fingerprinting and transcriptome analysis demonstrated the presence of putative glycosyl hydrolase and glucose oxidase (GOx) enzymes. These activities had been described earlier as parts of the fungus´ halogenation machinery, as they provide CPO with the co-substrate H2O2. The GOx activity was found to have a pH optimum of 5. Compared to the wild type values, GOx activity and glucose-driven MCD chlorination activity in the culture of a white mutant were found to be strongly increased to values of 1-2 U mL-1. As most CPO-catalyzed peroxidation reactions also show pH optima at around 5, the C. fumago culture supernatant can provide a highly convenient CPO/GOx source for many reactions with in situ H2O2 production.

Total Synthesis Establishes the Biosynthetic Pathway to the Naphterpin and Marinone Natural Products.

The naphterpins and marinones are naphthoquinone meroterpenoids with an unusual aromatic oxidation pattern that is biosynthesized from 1,3,6,8-tetrahydroxynaphthalene (THN). We propose that cryptic halogenation of THN derivatives by vanadium-dependent chloroperoxidase (VCPO) enzymes is key to this biosynthetic pathway, despite the absence of chlorine in these natural products. This speculation inspired a total synthesis to mimic the naphterpin/marinone biosynthetic pathway. In validation of this biogenetic hypothesis, two VCPOs were discovered that interconvert several of the proposed biosynthetic intermediates.

Spectroscopic and QM/MM investigations of Chloroperoxidase catalyzed degradation of orange G.

Chloroperoxidase (CPO), a heme-thiolate protein, from Caldariomyces fumago catalyzes a plethora of reactions including halogenation, dismutation, epoxidation, and oxidation. Although all CPO-catalyzed reactions go through a common intermediate, compound I, different mechanisms are followed in subsequent transformations. To understand the mechanism of CPO-catalyzed halide-dependent degradation of orange G, the role of halide and pH was systematically investigated. It is revealed that formation and protonation of compound X, a long-sought after hypochlorite heme adduct intermediate existed during CPO-catalyzed halide-dependent reactions, significantly lowers the reaction barrier and increases the efficiency of CPO-catalyzed orange G degradation. The extremely acidic optimal reaction pH suggests the protonation of a residue, presumably, Glu 183 in CPO catalysis. Halide dependent studies showed that Kcat is higher in the presence of Br(-) than in the presence of Cl(-). The degradation products of orange G indicate the cleavage at a single position of orange G, demonstrating a high regioselectivity of CPO-catalyzed degradation. Based on our kinetic, NMR and QM/MM studies, the mechanism of CPO-catalyzed orange G degradation was proposed.

Rapid and efficient degradation of bisphenol A by chloroperoxidase from Caldariomyces fumago: product analysis and ecotoxicity evaluation of the degraded solution.

OBJECTIVES: To degrade enzymatically bisphenol A (BPA) that causes serious environmental concerns and is difficult to be degraded by chemical or physical methods. RESULTS: BPA (150 mg l(-1)) was completely degraded by chloroperoxidase (CPO)/H2O2 within 7 min at room temperature, atmospheric pressure with the enzyme at 6 µg CPO ml(-1). The degradation products were identified by HPLC-MS, which suggested involvement of multiple steps. Enzymatic treatment followed by existing bioremediation technologies (activated sludge) enhanced removal of COD from 9 to 54 %. Using an ecotoxicity evaluation with Chlorella pyrenoidosa, the degradation products had a lower toxicity than BPA. CONCLUSION: BPA can be degraded rapidly and efficiently under mild conditions with chloroperoxidase at 6 µg ml(-1). The degradation products had a lower toxicity than BPA.

How the Proximal Pocket May Influence the Enantiospecificities of Chloroperoxidase-Catalyzed Epoxidations of Olefins.

Chloroperoxidase-catalyzed enantiospecific epoxidations of olefins are of significant biotechnological interest. Typical enantiomeric excesses are in the range of 66%-97% and translate into free energy differences on the order of 1 kcal/mol. These differences are generally attributed to the effect of the distal pocket. In this paper, we show that the influence of the proximal pocket on the electron transfer mechanism in the rate-limiting event may be just as significant for a quantitatively accurate account of the experimentally-measured enantiospecificities.

5¹V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site.

Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains "spectroscopically silent" V(V), which does not change oxidation state during the reaction. We employed an NMR crystallography approach based on (51)V magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO). The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5-100-fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained due to the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated at pH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and mutant VCPO and their pH-dependence. This study demonstrates that (51)V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.

Formation of chloroform and tetrachloroethene by Sinorhizobium meliloti strain 1021.

UNLABELLED: The mechanisms and organisms involved in the natural formation of volatile organohalogen compounds (VOX) are largely unknown. We provide evidence that the common and widespread soil bacterium Sinorhizobium meliloti strain 1021 is capable of producing up to 3338·6 ± 327·8 ng l(-1) headspace volume of chloroform (CHCl3 ) and 807·8 ± 13·5 ng l(-1)  headspace volume of tetrachloroethene (C2 Cl4 ) within 1 h when grown in soil extract medium. Biotic VOX formation has been suggested to be linked to the activity of halogenating enzymes such as haloperoxidases. We tested if the observed VOX formation by S. meliloti can be attributed to one of its chloroperoxidases (Smc01944) that is highly expressed in the presence of H2 O2. However, addition of 10 mmol l(-1) H2 O2 to the S. meliloti cultures decreased VOX formation by 52% for chloroform and 25% for tetrachloroethene, while viable cell numbers decreased by 23%. Interestingly, smc01944 gene expression increased 450-fold. The quantification of extracellular chlorination activity in cell suspension experiments did not provide evidence for a role of S. meliloti chloroperoxidases in the observed VOX formation. This suggests that a momentarily unknown mechanism which requires no H2 O2 might be responsible for the VOX formation by S. meliloti. Regardless of the underlying mechanism our results suggest that the soil bacterium S. meliloti might be an important source of VOX in soils. SIGNIFICANCE AND IMPACT OF THE STUDY: Volatile organohalogen compounds (VOX) strongly influence atmospheric chemistry and Earth's climate. Besides anthropogenic emissions they are naturally produced by either abiotic or biotic pathways in various environments. Particularly in soils, microbial processes drive the natural halogen cycle but the direct link to microbial VOX formation has not been studied in detail yet. In this study we provide evidence that the common and widespread soil bacterium Sinorhizobium meliloti strain 1021 forms chloroform and tetrachloroethene. The potential contribution of S. meliloti to soil VOX release could significantly influence soil and atmospheric chemistry.

Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor.

A novel nonheme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of Rhodococcus sp. strain ECU1013. RhEst1 was overexpressed in Escherichia coli BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The Km values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into (S)-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess (ee) of 97.5%, indicating an extremely high enantioselectivity (E = 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals.