Haloperoxidase Enzymes Research Articles

Insights into the Mechanism of a Vanadium Bromoperoxidase from the Marine Macro‐Algae Corallina pilulifera for Biocatalytic Halogenation

AbstractVanadium haloperoxidases have been studied to understand their mechanism and halide specificity. Crystal structures of vanadium bromoperoxidase enzyme complexes from Corallina piluifera, with vanadate and bromide and with phosphate and chloride, show significant displacement of loop residues 336–338 upon halogen binding. This shows a “closed position” of Leu337 locking the bromide ion in a hydrophobic environment favoring the vanadium peroxide reaction with the halogen by retaining the resulting hypobromite in the enzyme active site. The bound cofactor exists as a mixture of free vanadate and histidine553‐vanadate adduct. A mutant enzyme Arg397Trp also has been crystallized with bound phosphate and its structure determined with and without the bound bromide ion. The precise positions of the bromine have been determined using its anomalous signal. The bromide binding site in the mutant enzyme is displaced by 2.5 Å resulting in a mixed population of the “open” and the “closed” forms of Leu337. This allows additional chloroperoxidase activity due to re‐positioning of the halogen ion 0.6 Å closer to the vanadate ion. These studies support the application of vanadium haloperoxidase enzymes for selective halogenation of important drug molecules.

Ceria nanoparticles immobilized with self-assembling peptide for biocatalytic applications.

Peptide-based artificial enzymes exhibit structure and catalytic mechanisms comparable to natural enzymes but they suffer from limited reusability due to their existence in homogenous solutions. Immobilization of self-assembling peptides on the surface of nanoparticles can be used to overcome limitations associated with artificial enzymes. A high, local density of peptides can be obtained on nanoparticles to exert cooperative or synergistic effects, resulting in an accelerated rate of reaction, distinct catalytic properties, and excellent biocompatibility. In this work, we have immobilized a branched, self-assembled, and nanofibrous catalytic peptide, (C12-SHD)2KK(Alloc)-NH2, onto thiolated ceria nanoparticles to generate a heterogeneous catalyst with an enhanced number of catalytic sites. This artificial enzyme mimics the activities of esterase, phosphatase, and haloperoxidase enzymes and the catalytic efficiency remains nearly unaltered when reused. The enzyme-mimicking property is investigated for pesticide detection, bone regeneration, and antibiofouling applications. Overall, this work presents a facile approach to develop a multifunctional heterogeneous biocatalyst that addresses the challenges associated with unstable peptide-based homogeneous catalysts and, thus, shows a strong potential for industrial applications.

Generation of Ru(III)-hypochlorite with resemblance to the heme-dependent haloperoxidase enzyme.

The reaction of [(Me/BnTPEN)RuII(NCCH3)]2+ (BnTPEN = N1-benzyl-N1,N2,N2-tris(pyridine-2-ylmethyl)ethane-1,2-diamine and MeTPEN = N1-methyl-N1,N2,N2-tris(pyridine-2-ylmethyl)ethane-1,2-diamine) with mCPBA in the presence of chloride ions in CH3CN : H2O generated a novel (Me/BnTPEN)RuIII-OCl species at room temperature. This hypochlorite adduct could also be obtained by the direct reaction of NaOCl and HClO4 with (L)RuII complexes. The current study mimics the synthesis of a metal hypochlorite adduct in a similar fashion as in the heme-dependent haloperoxidase enzyme. As an electrophilic oxidant, the ruthenium hypochlorite adduct catalyzes hydrogen atom abstraction reactions of phenols and their derivatives.

High-throughput synthesis of CeO2 nanoparticles for transparent nanocomposites repelling Pseudomonas aeruginosa biofilms

Preventing bacteria from adhering to material surfaces is an important technical problem and a major cause of infection. One of nature’s defense strategies against bacterial colonization is based on the biohalogenation of signal substances that interfere with bacterial communication. Biohalogenation is catalyzed by haloperoxidases, a class of metal-dependent enzymes whose activity can be mimicked by ceria nanoparticles. Transparent CeO2/polycarbonate surfaces that prevent adhesion, proliferation, and spread of Pseudomonas aeruginosa PA14 were manufactured. Large amounts of monodisperse CeO2 nanoparticles were synthesized in segmented flow using a high-throughput microfluidic benchtop system using water/benzyl alcohol mixtures and oleylamine as capping agent. This reduced the reaction time for nanoceria by more than one order of magnitude compared to conventional batch methods. Ceria nanoparticles prepared by segmented flow showed high catalytic activity in halogenation reactions, which makes them highly efficient functional mimics of haloperoxidase enzymes. Haloperoxidases are used in nature by macroalgae to prevent formation of biofilms via halogenation of signaling compounds that interfere with bacterial cell–cell communication (“quorum sensing”). CeO2/polycarbonate nanocomposites were prepared by dip-coating plasma-treated polycarbonate panels in CeO2 dispersions. These showed a reduction in bacterial biofilm formation of up to 85% using P. aeruginosa PA14 as model organism. Besides biofilm formation, also the production of the virulence factor pyocyanin in is under control of the entire quorum sensing systems P. aeruginosa. CeO2/PC showed a decrease of up to 55% in pyocyanin production, whereas no effect on bacterial growth in liquid culture was observed. This indicates that CeO2 nanoparticles affect quorum sensing and inhibit biofilm formation in a non-biocidal manner.

Spectroscopic characterization of a Ru(III)-OCl intermediate: a structural mimic of haloperoxidase enzymes.

Haloperoxidase enzymes utilize metal hypohalite species to halogenate aliphatic and aromatic C-H bonds to C-X (X = Cl, Br, I) in nature. In this work, we report the synthesis and spectroscopic characterization of a unique RuIII-OCl species as a structural mimic of haloperoxidase enzymes. The reaction of [(BnTPEN)RuII(NCCH3)]2+ (BnTPEN = N1-benzyl-N1,N2,N2-tris(pyridine-2-ylmethyl)ethane-1,2-diamine) with hypochlorite in the presence of an acid in CH3CN : H2O mixtures generated a novel [(BnTPEN)RuIII-OCl]2+ species that persists for 4.5 h at room temperature. This new species was characterized by UV-vis absorption, EPR, and resonance Raman spectroscopic techniques, and ESI-MS. The RuIII-OCl species is capable of performing oxygen atom transfer and hydrogen atom abstraction to various organic substrates.

Defect-controlled halogenating properties of lanthanide-doped ceria nanozymes.

Marine organisms combat bacterial colonization by biohalogenation of signaling compounds that interfere with bacterial communication. These reactions are catalyzed by haloperoxidase enzymes, whose activity can be emulated by nanoceria using milli- and micromolar concentrations of Br- and H2O2. We show that the haloperoxidase-like activity of nanoceria can greatly be enhanced by Ln substitution in Ce1-xLnxO2-x/2. Non-agglomerated nanosized Ce1-xLnxO2-x/2 (Ln = Pr, Tb, particle size < 10 nm) was prepared mechanochemically from CeCl3 and Na2CO3 followed by short calcination. Lanthanide metals could be incorporated into the CeO2 host without solubility limit, as shown for Tb. The distribution of the Ln3+ defect sites in the CeO2 host structure was analyzed by electron spin resonance spectroscopy. Ce3+ and superoxide O2- species are present at surface sites. Their formation is promoted by increasing dopant concentration. Ce1-xLnxO2-x/2 was prepared in copious amounts by ball-milling. This energy-saving and residue-free method can be upscaled to industrial scale. The surface defect chemistry of Ce1-xLnxO2-x/2 was unravelled by vibrational spectroscopy. It is associated with the mechanochemical preparation and leads to enhanced catalytic activity. Although Ce0.9Pr0.1O1.95 had a lower BET surface area than pure CeO2, its catalytic activity, calibrated by oxidative bromination of phenol red, was much higher because the ζ-potential increased from 15 mV (for CeO2) to 30 mV (for Ce0.9Pr0.1O1.95). This facilitates adsorption of Br- in aqueous conditions and explains the high catalytic activity of the Ln-substituted CeO2. Ce1-xLnxO2-x/2 is an effective and "green" nanoparticle haloperoxidase mimic for antifouling applications, as no chemicals other than the ubiquitous Br- and H2O2 (generated in daylight) are required, and only natural metabolites are released into the environment.

Genetic and Biochemical Reconstitution of Bromoform Biosynthesis in Asparagopsis Lends Insights into Seaweed Reactive Oxygen Species Enzymology.

Marine macroalgae, seaweeds, are exceptionally prolific producers of halogenated natural products. Biosynthesis of halogenated molecules in seaweeds is inextricably linked to reactive oxygen species (ROS) signaling as hydrogen peroxide serves as a substrate for haloperoxidase enzymes that participate in the construction these halogenated molecules. Here, using red macroalga Asparagopsis taxiformis, a prolific producer of the ozone depleting molecule bromoform, we provide the discovery and biochemical characterization of a ROS-producing NAD(P)H oxidase from seaweeds. This discovery was enabled by our sequencing of Asparagopsis genomes, in which we find the gene encoding the ROS-producing enzyme to be clustered with genes encoding bromoform-producing haloperoxidases. Biochemical reconstitution of haloperoxidase activities establishes that fatty acid biosynthesis can provide viable hydrocarbon substrates for bromoform production. The ROS production haloperoxidase enzymology that we describe here advances seaweed biology and biochemistry by providing the molecular basis for decades worth of physiological observations in ROS and halogenated natural product biosyntheses.

Computational Study on the Catalytic Reaction Mechanism of Heme Haloperoxidase Enzymes

AbstractHeme haloperoxidases are unique enzymes in biology that react H2O2 and halides on a heme center to generate hypohalide, which reacts with a substrate by halide transfer. We studied model complexes of the active site of heme haloperoxidase and investigated the reaction mechanism starting from an iron(III)‐hydrogen peroxide‐heme complex. We find two stepwise proton transfers by active site Glu and His residues to form Compound I and water, whereby the second proton transfer step is rate‐determining. In a subsequent reaction with chloride the oxygen atom transfer is studied to form hypohalide. Overall, the free energy of activation of the second proton transfer and oxygen atom transfer to halide are similar in energy with free energies of activation of around 20 kcal mol−1. The calculations show that during oxygen atom transfer from Compound I to halide, significant charge‐transfer happens prior to the transition state. This implies that the reaction may be enhanced in polar environments and through second‐coordination sphere effects. The studies show that the conversion of H2O2 and halide on a heme center is fast and few intermediates along the reaction mechanism will have a lifetime that is long enough to enable trapping and characterization with experimental methods. A range of active site models and density functional theory methods were tested, but little effect is seen on the mechanism and optimized geometries.

Fluorescent Detection of Bromoperoxidase Activity in Microalgae and Planktonic Microbial Communities Using Aminophenyl Fluorescein

Among planktonic communities haloperoxidase enzymes may play a role in the control of intracellular and extracellular reactive oxygen species, in the generation of halogenated organic compounds and in chemical interactions between microbes. We introduce a sensitive fluorometric assay with a large dynamic range that is based on the dearylation of aminophenyl fluorescein (APF) to fluorescein by highly reactive oxygen species. Bromoperoxidase and chloroperoxidase enzymes catalyze the reaction between hydrogen peroxide and halides to generate highly reactive hypohalite intermediates able to dearylate APF. The fundamentals and standardization of the approach are illustrated using a partially purified, vanadium-dependent bromoperoxidase from the red seaweed Corallina officinalis. Laboratory cultures of two polar diatoms, Porosira glacialis and Fragilariopsis cylindrus, are used to illustrate the sensitivity and potential applications of the approach for in vitro, in vivo and in situ measurements of bromoperoxidase activity. These two diatoms differ in biovolume-specific bromoperoxidase activity by 2-orders of magnitude, from 5.4 to 0.044 fmol fluorescein μm-3 h-1, respectively. The approach is also used to investigate the partition of haloperoxidase activity between different size fractions of summer coastal planktonic communities, illustrating that generally more than 50 % of the haloperoxidase activity occurred in a > 10 μm size fraction that was dominated by diatoms. The assay has the potential to be of value in many aspects of haloperoxidase research, including developing an improved understanding of the roles of haloperoxidase enzymes in microbial planktonic communities.

Coordination environment changes of the vanadium in vanadium-dependent haloperoxidase enzymes

Vanadium-dependent haloperoxidases are a class of enzymes that catalyze oxidation reactions with halides to form halogenated organic products and water. These enzymes include chloroperoxidase and bromoperoxidase, which have very different protein sequences and sizes, but regardless the coordination environment of the active sites is surprisingly constant. In this manuscript, the comparison of the coordination chemistry of V-containing-haloperoxidases of the trigonal bipyramidal geometry was done by data mining. The catalytic cycle imposes changes in the coordination geometry of the vanadium to accommodate the peroxidovanadium(V) intermediate in an environment we describe as a distorted square pyramidal geometry. During the catalytic cycle, this intermediate converts to a trigonal bipyramidal intermediate before losing the halogen and forming a tetrahedral vanadium-protein intermediate. Importantly, the catalysis is facilitated by a proton-relay system supplied by the second sphere coordination environment and the changes in the coordination environment of the vanadium(V) making this process unique among protein catalyzed processes. The analysis of the coordination chemistry shows that the active site is very tightly regulated with only minor changes in the coordination geometry. The coordination geometry in the protein structures deviates from that found for both small molecules crystalized in the absence of protein and the reported functional small molecule model compounds. At this time there are no examples reported of a structurally similar small molecule with the geometry observed for the peroxidovanadium(V) in the active site of the vanadium-containing haloperoxidases.

Two dinuclear oxidovanadium(V) complexes of N2O2 donor amine-bis(phenolate) ligands with bromo-peroxidase activities: Kinetic, catalytic and computational studies

Two dinuclear oxidovanadium(V) complexes [LiVVO(µ2-O)VVO(Li)] (i = 1, H2L1, complex 1 and i = 2 for H2L2, complex 2) of two ONNO donor amine-bis(phenolate) ligands have been synthesized and characterized by X-ray diffraction studies which exhibited distorted octahedral geometry around each V center. In MeCN the complexes exist as dimers as indicated by HRMS studies, however, in the presence of 2 or more equivalents of H+ the dimers turned into monomers, ([LiVV = O]+ which exists in equilibrium with ([LiVV = OH]2+ and evidenced from the shift in λmax from 685 nm to 765 nm for complex 1 and 600 to 765 nm for complex 2. The complexes 1 and 2 efficiently catalyze the oxidative bromination of salicylaldehyde in the presence of H2O2 to produce 5-bromo-salicylaldehyde as the major product with TONs 405 and 450, respectively in the mixed solvent system (H2O:MeOH:THF = 4:3:2, v/v). The kinetic analysis of the bromide ion oxidation reaction indicates a mechanism which is first order in peroxidovanadium complex and bromide ion and limiting first-order on [H+]. The evaluated kBr and kH values are (8.82 ± 0.35) and (65.0 ± 2.23) M−1 s−1 for complex 1 and (6.74 ± 0.19) and (61.87 ± 2.27) M−1 s−1 for complex 2, respectively. The Ka of protonated species ([LiVV = OH]2+ are: Ka = (4.3 ± 0.40) × 10−3 (pKa = 2.37) and (4.7 ± 0.50) × 10−3 (pKa = 2.33) for complex 1 and 2 respectively. On the basis of the chemistry displayed by these model compounds, a mechanism of bromide oxidation and a tentative catalytic cycle have been framed which might be relevant to vanadium haloperoxidase enzymes and supported by DFT calculations.

Experimental Study and Modeling of the UV-Vis and Infrared Spectra of the [VO(O2)Hheida]- Complex Dissolved in Water.

Combined theoretical and experimental studies of the [VO(O2)Hheida]- anion dissolved in water that may serve as a functional model for vanadium haloperoxidase enzymes have been performed. The geometrical structure and absorption and vibrational spectra of this system have been evaluated within the framework of density functional theory (DFT). The obtained theoretical results on the equilibrium structure and optical spectra are in quite good agreement with the experimental data. With the aid of the combination of UV-visible spectroscopy and electronic structure calculations, it has been revealed that, in the apparent absorption spectra of the [VO(O2)Hheida]- anion, the highest in energy band corresponds to a ligand to metal electron excitation, while the band with a maximum at 430 nm arises from the peroxo group. The calculations also reproduce quite well the positions, intensities and the grouping of frequencies in the near-infrared (NIR) spectra. The visualization of the calculated vibrations in the energy range of 400-1100 cm-1 has been presented.

The Use Haloperoxidases in Organic Synthesis: Selected Reactions of Oxidation, Epoxydation and Sulfoxidation

&lt;p&gt;Haloperoxidases are ubiquitous metalloenzymes that catalyse a variety of enantioselective oxygen-transfer reactions with hydrogen peroxide or alkylperoxides. Haloperoxidases are enzymes which catalyze the reaction of oxidation, epoxidation and sulfoxidation by hydrogen peroxide. These enzymes usually contain the FeHeme moiety or vanadium as an essential constituent at their active site, however, a few haloperoxidases which lack a metal cofactor are known. This review will examine the reactivity of the different haloperoxidases, particularly the mechanism of oxidation by hydrogen peroxide, and the mechanism of oxidation and sulfoxidation, including the newly reported regioselectivity and enantioselectivity of the haloperoxidases. The structure of chloroperoxidase, the vanadium active site and the role of critical amino acid side chains for catalysis and functional biomimetic systems, with specific relevance to the mechanism of the haloperoxidase enzymes. Advances have recently been made in using them to prepare, under controlled conditions, chiral organic molecules that are valuable for the synthesis of a wide range of useful compounds. The application of biocatalytic methods in asymmetric organic synthesis is of great interest as an alternative to chemical procedures employing chiral auxiliaries. Asymmetric oxidation of prochiral sulfides to yield optically active sulfoxides has been performed by many different techniques yielding varying enantiomeric excess values. Oxygenated metabolites are compounds that are commonly found in nature and they are produced by many different organisms. The oxygen atom is incorporated into organic compounds by enzyme-catalyzed reactions with oxygen ions as the oxygen source. For over 40 years haloperoxidases were thought to be responsible for the incorporation of mainly halogen atoms into organic molecules. However, haloperoxidases lack substrate specificity and regioselectivity, and the connection of haloperoxidases with the in vivo formation of oxygenated as well as halometabolites has been demonstrated. Recently, molecular genetic investigations showed that, at least in bacteria, fungi, and other organisms a different class of halogenases is involved in halo- and oxygenated metabolite formation. These halogenases were found to require FADH2, which can be produced from FAD and NADH by unspecific flavin reductases. The FADH&lt;sub&gt;2&lt;/sub&gt;-dependent halogenases and haloperoxidases show substrate specificity and regioselectivity, and their genes have been detected in many halometabolite-producing organisms, suggesting that this type of halogenating enzymes constitutes the major source for halo- and oxygenated metabolite formation in bacteria and also in other organisms. Distribution of haloperoxidases in nature also is demonstrated in this brief review.&lt;/p&gt;

DFT Studies and Experiments on Biocatalytic Centers: Structure, Vibrations, and Core Excitations of the K[VO(O2)Hheida] Complex

Geometric and electronic properties of the K[VO(O2)Hheida] complex, which serves as a functional model for vanadium haloperoxidase enzymes, as well as of the molecular ions (VO(O2)Hheida)− and (VO(O)Hheida)− are evaluated using density-functional theory (DFT). Theoretical results of equilibrium structures, vibrational excitations, oxygen 1s core ionization and excitation are compared with measured data. The theoretical equilibrium structure of the K[VO(O2)Hheida] complex agrees quite well with the corresponding molecular structure in crystalline K[VO(O2)Hheida]·2(H2O) and differs only a little from that of the (VO(O2)Hheida)− ion. The potassium appears in the K[VO(O2)Hheida] complex as a positive K+ species binding only electrostatically with no orbital hybridization to the (VO(O2)Hheida)− part, which is also obvious from the respective orbital analyses and densities-of-states. The vibrational modes of peroxo and vanadyl oxygen in K[VO(O2)Hheida] are strongly coupled, and the calculated excitation energies can explain details of the experimental infrared and Raman spectra. The theoretical O 1s core ionization potentials (IP) vary between the different oxygen species and are consistent with results from X-ray photoemission (XPS). Theoretical O 1s core excitation spectra are confirmed by results from O K-edge NEXAFS measurements for crystalline K[VO(O2)Hheida]·2(H2O) under oxygen and helium pressure. The difference between the two experimental spectra can be explained by the presence of oxygen-deficient species based on the theoretical findings for the (VO(O)Hheida)− species.

The Vanadium Iodoperoxidase from the marine flavobacteriaceae species Zobellia galactanivorans reveals novel molecular and evolutionary features of halide specificity in the vanadium haloperoxidase enzyme family.

Vanadium haloperoxidases (VHPO) are key enzymes that oxidize halides and are involved in the biosynthesis of organo-halogens. Until now, only chloroperoxidases (VCPO) and bromoperoxidases (VBPO) have been characterized structurally, mainly from eukaryotic species. Three putative VHPO genes were predicted in the genome of the flavobacterium Zobellia galactanivorans, a marine bacterium associated with macroalgae. In a phylogenetic analysis, these putative bacterial VHPO were closely related to other VHPO from diverse bacterial phyla but clustered independently from eukaryotic algal VBPO and fungal VCPO. Two of these bacterial VHPO, heterogeneously produced in Escherichia coli, were found to be strictly specific for iodide oxidation. The crystal structure of one of these vanadium-dependent iodoperoxidases, Zg-VIPO1, was solved by multiwavelength anomalous diffraction at 1.8 Å, revealing a monomeric structure mainly folded into α-helices. This three-dimensional structure is relatively similar to those of VCPO of the fungus Curvularia inaequalis and of Streptomyces sp. and is superimposable onto the dimeric structure of algal VBPO. Surprisingly, the vanadate binding site of Zg-VIPO1 is strictly conserved with the fungal VCPO active site. Using site-directed mutagenesis, we showed that specific amino acids and the associated hydrogen bonding network around the vanadate center are essential for the catalytic properties and also the iodide specificity of Zg-VIPO1. Altogether, phylogeny and structure-function data support the finding that iodoperoxidase activities evolved independently in bacterial and algal lineages, and this sheds light on the evolution of the VHPO enzyme family.

Biocatalytic formation of synthetic melanin: The role of vanadium haloperoxidases, L-DOPA and iodide

The vanadium haloperoxidase (V-HPO) enzyme, extracted from the brown alga Laminaria saccharina, is able to catalyze the formation of a black precipitate, using as precursor the amino acid l-dopa in the presence of hydrogen peroxide and iodide, in one-pot synthesis. The l-dopa oxidation is a multistep reaction with a crucial role played by the iodide in the enzyme catalyzed peroxidative production of dopachrome, a well known intermediate in the synthesis of melanin. Dopachrome is then converted to a synthetic form of melanin through a polymerization reaction. Factors, such as buffer composition and pH, influence significantly the reaction first steps, but further steps of melanin production are hardly influenced. The biosynthetic melanin produced through the combination V-HPO/I/H 2O 2, was characterized by several spectroscopic techniques (UV–vis and FT-IR) as well as XRD. Moreover, this biopolymer is light sensitive, decomposing into oligo- and monomeric units. Scanning electron microscopy (SEM) imaging showed different morphologies when compared with commercial available melanin. The biosynthetic production of melanin can have a wide range of applications from photosensitive cells to biomedicine with the advantage of being produced under eco-friendly and mild conditions.

ChemInform Abstract: Monoperoxidovanadium(V) Complexes: Synthesis, Structure and Solution Properties

AbstractReview: 89 refs.

Effects of hydrogen peroxide on the content of major volatile halogenated compounds in the red alga Asparagopsis taxiformis (Bonnemaisoniaceae)

The genus Asparagopsis is a prolific source of halogenated metabolites. Due to its commercial applications, it has been intensively cultivated in southern Portugal. In the present study, we assess if the internal levels of the major halogenated metabolites (bromoform and dibromoacetic acid) in Asparagopsis taxiformis can be increased with hydrogen peroxide (H2O2) addition. Previous studies with red algae showed that the production/release of bromoform can be enhanced by exogenously supplying H2O2. However, no study has assessed if H2O2 supply enhances the content of secondary metabolites within the biomass. This detail is important as the objective of the proposed research is to enhance the content of these valuable metabolites in the produced biomass. Both the activity of the haloperoxidase enzyme and the metabolite content were assessed on short-term and long-term incubation periods to H2O2. To determine the susceptibility of A. taxiformis photosynthetic performance to the imposed oxidative stress, the in vivo fluorescence of photosystem II was monitored. A. taxiformis was shown to be physiologically vulnerable to H2O2, given the observed decrease of the maximum quantum yield of photosynthesis (F v/F m). Contrary to what was expected, the presence of H2O2 inhibited the activity of the iodoperoxidase enzyme. Nevertheless, the extracted halogenated metabolites were higher over the first hours of exposure to H2O2, decreasing after 48 h. These results are probably related to the prosthetic group of the halogenated enzyme in A. taxiformis and the long-term oxidative stress damage of H2O2 exposure. Considering the objective of the proposed research, addition of H2O2 to the cultures, prior (3 h) to biomass harvesting, increases the metabolite content.

Presence of Surface Vanadium Peroxo-oxo Umbrella Structures in Supported Vanadium Oxide Catalysts: Fact or Fiction?

Recent debates over the active site structure of supported vanadia/silica catalysts have suggested the existence of a surface vanadium peroxo-oxo "umbrella" structure. This study definitively demonstrates with Raman and UV-vis spectroscopy that the surface vanadia peroxo-oxo umbrella-like structure is not present for both hydrated and dehydrated supported vanadia catalysts such as supported vanadia on silica. The vanadia peroxo-oxo umbrella structure, however, is present in vanadium haloperoxidase (VHPOs) enzymes and metal-organic compounds designed to mimic VHPOs.

Red seaweed enzyme‐catalyzed bromination of bromophenol red: An inquiry‐based kinetics laboratory experiment for undergraduates

Haloperoxidase enzymes are of interest for basic and applied bioscientists because of their increasing importance in pharmaceutical industry and environmental cleanups. In a guided inquiry-based laboratory experiment for life-science, agricultural science, and health science undergraduates, the bromoperoxidase from a red seaweed was used to brominate bromophenol red, a novel starting substrate. Substrate and enzyme concentration dependence of this enzyme-catalyzed reaction as followed colorimetrically yielded initial rates of reaction that the students compared with those of their peers. The students worked as collaborative groups partially designing their own experiments and carrying them out. In performing the laboratory experiment, they were minimally guided by the instructor and teaching assistants. To engage the students before the laboratory, a short activity involving the enzyme-induced color change was carried out. At the end of the laboratory session, student groups discussed their results in front of the class and reached their own conclusions. Most students had better understanding of important concepts in enzyme kinetics and showed good attitude toward the overall student-centered laboratory exercise.