Ferric Enzyme Research Articles

Spectroscopic definition of ferrous active sites in non-heme iron enzymes.

Non-heme iron enzymes play key roles in antibiotic, neurotransmitter, and natural product biosynthesis, DNA repair, hypoxia regulation, and disease states. These enzymes had been refractory to traditional bioinorganic spectroscopic methods. Thus, we developed variable-temperature variable-field magnetic circular dichroism (VTVH MCD) spectroscopy to experimentally define the excited and ground ligand field states of non-heme ferrous enzymes (Solomon et al., 1995). This method provides detailed geometric and electronic structure insight and thus enables a molecular level understanding of catalytic mechanisms. Application of this method across the five classes of non-heme ferrous enzymes has defined that a general mechanistic strategy is utilized where O2 activation is controlled to occur only in the presence of all cosubstrates.

Resonance Raman of the nitrite reductase action of heme‐copper oxidoreductases

Several themes have emerged in the studies of biological control of nitrite and nitric‐oxide chemistry.[1‐2] Apparently, evolution has clearly selected means by which to take advantage of the unique NO2 and NO chemistry, and a number of enzymes have evolved that are able to activate NO2/NO kinetically, and control their redox chemistry. In bacterial respiration the reduction of NO2/NO to N2O by terminal oxidoreductases supports the hypothesis of a common evolutionary origin of bacterial denitrification and aerobic respiration. Comparison of the enzymes responsible for the activation of NO in denitrification and bacterial respiration may provide the means to identify conserved structural features, which can be assumed to be involved in basic functions common to both classes of enzymes. The most commonly encountered metals in the biological reduction of NO2 to NO and subsequently to N2O are iron and copper in mononuclear, binuclear, and dinuclear centers. In P450Nor the electrons needed for the reduction of NO are directly transferred from NADH. This way, a single molecule of NO binds at the heme Fe, and addition of two electrons to heme Fe3+‐NO yields the two electron reduced species Fe2+‐N=O‐. A second NO moleculeattacks the N atom of the ferrous‐NO species to transiently yield hyponitrite (HONNO–), and thus the N‐N bond formation. Cleavage of the N‐O bond produces the ferric enzyme, N2O and H2O. Resonance Raman studies of the NO2‐ligand binding properties of heme‐copper oxidases will be presented.References. Discrete Ligand Binding and Electron Transfer Properties of ba3‐Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and …C Koutsoupakis, T Soulimane, C. Varotsis Accounts of chemical research 52 (5), 1380‐139, 2019. The structure of a ferrous heme‐nitro species in the binuclear heme a3/CuBcenter of ba3‐cytochrome c oxidase as determined by resonance Raman spectroscopy A. Loullis, MR Noor, T Soulimane, E Pinakoulaki Chemical Communications 51 (2), 286‐289, 2015.

To Be, or Not to Be, an Inhibitor: A Comparison of Azole Interactions with and Oxidation by a Cytochrome P450 Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from Rhodopseudomonas palustris HaA2, to compare how imidazolyl and triazolyl inhibitors bind to ferric and ferrous heme. 4-(Imidazol-1-yl)benzoic acid induced a red shift in the Soret wavelength (424 nm) in the ferric enzyme along with an increase and a decrease in the intensities of the δ and α bands, respectively. 4-(1H-1,2,4-Triazol-1-yl)benzoic acid binds to CYP199A4 with a 10-fold lower affinity and induces a smaller red shift in the Soret band. The crystal structures of CYP199A4 with these two inhibitors confirmed that these differences in the optical spectra were due to coordination of the imidazolyl ligand to the ferric Fe, but the triazolyl inhibitor interacts with, rather than displaces, the ferric aqua ligand. Additional water molecules were present in the active site of 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound CYP199A4. The space required to accommodate these additional water molecules in the active site necessitates changes in the position of the hydrophobic phenylalanine 298 residue. Upon reduction of the heme, the imidazole-based inhibitor Fe-N ligation was not retained. A 5-coordinate heme was also the predominant species in 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound ferrous CYP199A4, but there was an obvious shoulder at 447 nm indicative of some degree of Fe-N coordination. Rather than inhibit CYP199A4, 4-(imidazol-1-yl)benzoic acid was a substrate and was oxidized to generate a metabolite derived from ring opening of the imidazolyl ring: 4-[[2-(formylamino)acetyl]amino]benzoic acid.

Mechanisms of O2 Activation by Mononuclear Non-Heme Iron Enzymes.

Two major subclasses of mononuclear non-heme ferrous enzymes use two electron-donating organic cofactors (α-ketoglutarate or pterin) to activate O2 to form FeIV═O intermediates that further react with their substrates through hydrogen atom abstraction or electrophilic aromatic substitution. New spectroscopic methodologies have been developed, enabling the study of the active sites in these enzymes and their oxygen intermediates. Coupled to electronic structure calculations, the results of these spectroscopies provide fundamental insight into mechanism. This Perspective summarizes the results of these studies in elucidating the mechanism of dioxygen activation to form the FeIV═O intermediate and the geometric and electronic structure of this intermediate that enables its high reactivity and selectivity in product formation.

The metal- and substrate-dependences of 2,4′-dihydroxyacetophenone dioxygenase

While the enzyme, 2,4′-dihydroxyacetophenone dioxygenase (DAD), has been known for decades, very little has been characterized of the mechanism of the DAD-catalyzed oxidative cleavage of its reported substrate, 2,4′-dihydroxyacetophenone (DHA). The purpose of this study was to identify the active metal center and to characterize the substrate-dependence of the kinetics of the reaction to lay the foundation for deeper mechanistic investigation. To this, the DAD V1M mutant (bDAD) was overexpressed, purified, and reconstituted with various metal ions. Kinetic assays evaluating the activity of the reconstituted enzyme as well as the substrate- and product-dependences of the reaction kinetics were performed. The results from reconstitution of the apoprotein with a variety of metal ions support the requirement for an Fe3+ center for enzyme activity. Reaction rates showed simple saturation kinetics for DHA with values for kcat and KDHA of 2.4 s−1 and 0.7 μM, respectively, but no significant dependence on the concentration of O2. A low-level inhibition (KI = 1100 μM) by the 4HB product was observed. The results support a minimal kinetic model wherein DHA binds to resting ferric enzyme followed by rapid addition of O2 to yield an intermediate complex that irreversibly collapses to products.

Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate–dependent nonheme ferrous enzymes

Determining the requirements for efficient oxygen (O2) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O2) and a sequential mechanism (cofactor binding and reaction with O2 precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the FeII), αKG binding to FeII-DAOCS results in ∼45% five-coordinate sites that selectively react with O2 relative to the remaining six-coordinate sites. However, this reaction produces an FeIII species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to FeII-DAOCS produces five-coordinate sites that rapidly react with O2 to form an FeIV=O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes.

Effects of repetitive magnetic stimulation on the growth of primarily cultured hippocampus neurons in vitro and their expression of iron-containing enzymes.

Background: The mechanism of action of repetitive transcranial magnetic stimulation (rTMS) involves the generation of neuronal and action potentials utilizing induced currents in time-varying magnetic fields. However, the long-lasting and effective biological impact of magnetic stimulation does not appear to be completely explained by the transient magnetic field pulses. In this context, we hypothesized magnetic stimulation may affect the expression of iron-containing enzymes in neurons, mediating the long-lasting biological effects associated with this stimulus. Methods: Primarily cultured hippocampus neurons from SD rats were used as the cell model in this study. These were randomly divided into control, sham, and magnetic stimulation groups to probe into the effect of the magnetic field directly. The latter group received 40%, 60%, and 100% maximal stimulator output Tesla (1.68, 2.52, and 4.2 T) with low-frequency rTMS (1 Hz). The expression of iron-containing enzymes (catalase and aconitase) and non-ferrous enzymes (protein kinase A) was measured with Western blotting and ELISA. Results: The survival rates of neurons in the 40%T and 60%T groups were significantly increased in comparison to the controls (P<0.05), while those in the 100%T group showed cell damage, with slightly disturbed neurite connections and decreased survival rate. Furthermore, catalase and aconitase expression was higher in all of the stimulated groups in comparison to controls (P<0.05). On the other hand, the expression of the iron-containing enzymes decreased in the 100%T group in comparison with the 40%T and 60%T groups (P<0.05). Meanwhile, the expression of protein kinase A was not significantly increased in the groups which underwent magnetic stimulation. Conclusion: rTMS may increase the expression of ferrous enzymes but does not have a strong effect on non-ferrous enzymes. Excessive intensity of magnetic stimulation may reduce neuronal survival rate and affect the expression of iron-containing enzymes. The mechanism underlying the lasting effect of rTMS may be related to the increase of ferriferous expression induced by magnetic stimulation, with a clear correlation with stimulation intensity.

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5-Coordinate CO-Heme.

Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC-CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC-NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5-coordinate CO-heme, but the detailed mechanism remains unresolved. In this study, we examined the mechanism under the condition including BAY 41-2272, 2'-deoxy-3'-GMP and foscarnet. The addition of these effectors caused the original 6-coordinate CO-heme to convert to an end product that was an equimolar mixture of a 5- and a new 6-coordinate CO-heme, as assessed by IR spectral measurements. The two types of CO-hemes in the end product were further confirmed by CO dissociation kinetics. Stopped-flow measurements under the condition indicated that the ferrous sGC bound CO as two reversible steps, where the primary step was assigned to the full conversion of the ferrous enzyme to the 6-coordinate CO-heme, and subsequently followed by the slower second step leading a partial conversion of the 6-coordinate CO-heme to the 5-coordinate CO-heme. The observed rates for both steps linearly depended on CO concentrations. The unexpected CO dependence of the rates in the second step supports a multistep mechanism, in which the 5-coordinate CO-heme is led by CO release from a putative bis-carbonyl intermediate that is likely provided by the binding of a second CO to the 6-coordinate CO-heme. This mechanism provides a new aspect on the activation of sGC by CO.

Recruitment and Regulation of the Non-ribosomal Peptide Synthetase Modifying Cytochrome P450 Involved in Nikkomycin Biosynthesis.

The β-hydroxylation of l-histidine is the first step in the biosynthesis of the imidazolone base of the antifungal drug nikkomycin. The cytochrome P450 (NikQ) hydroxylates the amino acid while it is appended via a phosphopantetheine linker to the non-ribosomal peptide synthetase (NRPS) NikP1. The latter enzyme is comprised of an MbtH and single adenylation and thiolation domains, a minimal composition that allows for detailed binding and kinetics studies using an intact and homogeneous NRPS substrate. Electron paramagnetic resonance studies confirm that a stable complex is formed with NikQ and NikP1 when the amino acid is tethered. Size exclusion chromatography is used to further refine the principal components that are required for this interaction. NikQ binds NikP1 in the fully charged state, but binding also occurs when NikP1 is lacking both the phosphopantetheine arm and appended amino acid. This demonstrates that the interaction is mainly guided by presentation of the thiolation domain interface, rather than the attached amino acid. Electrochemistry and transient kinetics have been used to probe the influence of l-His-NikP1 binding on catalysis by NikQ. Unlike many P450s, the binding of substrate fails to induce significant changes on the redox potential and autoxidation properties of NikQ and slows down the binding of dioxygen to the ferrous enzyme to initiate catalysis. Collectively, these studies demonstrate a complex interplay between the NRPS maturation process and the recruitment and regulation of an auxiliary tailoring enzyme required for natural product biosynthesis.

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1

Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H2O2-mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV.

Dynamics of Lysine as a Heme Axial Ligand: NMR Analysis of the Chlamydomonas reinhardtii Hemoglobin THB1.

Nitrate metabolism in Chlamydomonas reinhardtii involves THB1, a monomeric hemoglobin thought to function as a nitric oxide dioxygenase (NOD). NOD activity requires dioxygen and nitric oxide binding followed by a one-electron oxidation of the heme iron and nitrate release. Unlike pentacoordinate flavohemoglobins, which are efficient NODs, THB1 uses two iron axial ligands: the conserved proximal histidine and a distal lysine (Lys53). As a ligand in both the oxidized (ferric) and reduced (ferrous) states, Lys53 is expected to lower the reorganization energy associated with electron transfer and therefore facilitate reduction of the ferric enzyme. In ferrous THB1, however, Lys53 must be displaced for substrate binding. To characterize Lys53 dynamics, THB1 was studied at various pH, temperatures, and pressures by NMR spectroscopy. Structural information indicates that the protein fold and Lys53 environment are independent of the oxidation state. High-pressure NMR experiments provided evidence that displacement of Lys53 occurs through fast equilibrium (∼3-4 × 103 s-1 at 1 bar, 298 K) with a low-population intermediate in which Lys53 is neutral and decoordinated. Once decoordinated, Lys53 is able to orient toward solvent and become protonated. The global lysine decoordination/reorientation/protonation processes measured by 15Nz-exchange spectroscopy are slow on the chemical shift time scale (101-102 s-1 at pH ≈ 6.5, 298 K) in both iron redox states. Thus, reorientation/protonation steps in ferrous THB1 appear to present a significant barrier for dioxygen binding, and consequently, NOD turnover. The results illustrate the role of distal ligand dynamics in regulating the kinetics of multistep heme redox reactions.

New function of enzyme involved in the formation of a carbon‐nitrogen triple bond

We have studied microbial metabolism of nitrile at the protein and gene levels. We discovered a nitrile‐synthesizing enzyme linked with nitrile hydratase (which is an industrial enzyme used for the production of amide from nitrile) in the nitrile metabolism. The nitrile‐synthesizing enzyme, which is involved in the synthesis of a carbon‐nitrogen triple bond, is a unique enzyme, which catalyzes the dehydration of aldoxime [R‐CH=N‐OH] into nitrile [R‐CN] even in an aqueous solution. This enzyme contains a heme as the prosthetic group. Unlike the utilization of H2O2 or O2 as a mediator of the catalysis by other heme‐containing enzymes, this enzyme is notable for the direct binding of a substrate to the heme ion. We previously reported that ferrous enzyme, containing a five‐coordinated high‐spin heme and a histidine residue as its proximal ligand, is the reactive form of the enzyme, and that another histidine in the distal heme pocket plays a crucial role in the catalysis. We determined the crystal structure of the enzyme and clarified the detailed reaction mechanism. Here, we for the first time found catalase activity of the enzyme, when 10 mM H2O2 was used as a substrate. We also determined kinetic parameters of this reaction. Based upon the three‐dimensional structure of this enzyme, we have tried to construct a mutated enzyme, which shows higher activity than the wild type enzyme.

(Invited) Molecular Mechanism of NO Reduction By Bacterial Nitric Oxide Reductases

Bacterial nitric oxide reductase (NOR) catalyzes the NO reduction reaction (2NO + 2H+ + 2e- --> N2O + H2O) at its active site, which is a binuclear center comprised by heme (heme b 3) and non-heme iron (FeB). In the enzymatic reaction by NOR, three short-lived intermediates have been proposed for the coordination of two molecules of NO to the binuclear center; i.e., trans-, cis-heme b 3- and cis-FeB-coordinations. To establish the molecular mechanism of the NO reduction by NOR, we have been studying the time-resolved spectroscopies to characterize the coordination and electronic structures of the intermediates, which are appeared in the micro- to milli-second time region after the reaction of the ferrous enzyme with the NO molecule(s). On the basis of the time-resolved optical, ESR and IR spectral changes, we have proposed that the trans-mechanism seem favorable for the NO reduction mechanism by bacterial NOR. We will discuss the NOR reaction with referring their crystallographic structural data which we reported.

Hydrogen peroxide-independent generation of superoxide catalyzed by soybean peroxidase in response to ferrous ion

It is well documented that extracellular alkalization occurs in plants under the challenges by pathogenic microbes. This may eventually induce the pH-dependent extracellular peroxidase-mediated oxidative burst at the site of microbial challenges. By employing the purified proteins of horseradish peroxidase as a model, we have recently proposed a likely role for free Fe2+ in reduction of ferric enzyme of plant peroxidases into ferrous intermediate and oxygen-bound form of enzyme known as Compound III which may eventually releases superoxide anion radical (O2•−), especially under alkaline condition, possibly contributing to the plant defense mechanism. In the present study, we employed the purified protein of soybean peroxidase (SBP) as an additional model, and examined the changes in the redox status of enzyme accompanying the generation of O2•− in response to Fe2+ under alkaline condition.

Enhancing hypothiocyanite production by lactoperoxidase – mechanism and chemical properties of promotors

BackgroundThe heme enzyme lactoperoxidase is found in body secretions where it significantly contributes to the humoral immune response against pathogens. After activation the peroxidase oxidizes thiocyanate to hypothiocyanite which is known for its microbicidal properties. Yet several pathologies are accompanied by a disturbed hypothiocyanite production which results in a reduced immune defense. MethodsThe results were obtained by measuring enzyme-kinetic parameters using UV–vis spectroscopy and a standardized enzyme-kinetic test system as well as by the determination of second order rate constants using stopped-flow spectroscopy. ResultsIn this study we systematically tested thirty aromatic substrates for their efficiency to promote the lactoperoxidase-mediated hypothiocyanite production by restoring the native ferric enzyme state. Thereby hydrophobic compounds with a 3,4-dihydroxyphenyl partial structure such as hydroxytyrosol and selected flavonoids emerged as highly efficient promotors of the (pseudo-)halogenating lactoperoxidase activity. ConclusionsThis study discusses important structure-function relationships of efficient aromatic LPO substrates and may contribute to the development of new agents to promote lactoperoxidase activity in secretory fluids of patients. SignificanceThis study may contribute to a better understanding of the (patho-)physiological importance of the (pseudo-)halogenating lactoperoxidase activity. The presented results may in future lead to the development of new therapeutic strategies which, by reactivating lactoperoxidase-derived hypothiocyanite production, promote the immunological activity of this enzyme.

The Dehaloperoxidase Paradox: How Nature Juggles Oxygen Transport, Peroxidase, and Peroxygenase Activities at a Single Heme Active Site

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is the first oxygen-binding globin that possesses a biologically relevant peroxidase activity. The two known isoforms of DHP, A and B, have been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that utilizes hydrogen peroxide as the oxidant. More recently, however, we have shown that DHP possesses both peroxygenase and oxidase activities using a new class of substrate, haloindoles, in addition to its previously identified O2-binding and peroxidase functions. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope 18O-labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2 18O2, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded 5,5’-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, and O2-concentration studies demonstrated dioxygen consumption. Non-enzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5’-Br2-indigo, and together suggest a newly identified oxidase activity for DHP. Preliminary data also suggest that DHP is able to catalyze the oxidation of 2,3-dimethylindole by a previously unreported oxygenase activity. The two major products were identified by HPLC and LC-MS analysis to be the dioxygenated product o-acetamidoacetophenone and the monooxygenated product 3-hydroxy-2,3-dimethylindole. Isotope labeling studies verified that the oxygen atom incorporation into the products were derived exclusively from molecular oxygen. Consistent with these results, the reaction did not proceed under anaerobic conditions, and was also decreased in the presence of radical scavengers. Our initial studies suggest that the observed radical-based oxygenase activity of DHP is reminiscent of lipoxygenase, a non-heme dioxygenase. The breadth of chemical reactivity demonstrated by DHP not only expands our current understanding of this multifunctional enzyme, but also highlights the plasticity of a single heme active site within a globin-fold to function as an oxygen-transport center, as a peroxidase for phenolic substrate oxidation, and as a peroxygenase for indolic substrate oxygenation. Figure 1

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapid-mixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pH-jump spectrophotometry revealed a basic pKa of 10.0 for the Fe(IV)-O-H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C-H and phenolic substrates, with second-order rate constants for C-H and O-H bond scission in the range of 10-10(7) M(-1)⋅s(-1). Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C-H and O-H bond scission.

A role for catalase-peroxidase large loop 2 revealed by deletion mutagenesis: control of active site water and ferric enzyme reactivity.

Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.

Antioxidant, Enzyme Inhibitory and Anti-Obesity Potential of Sorrel Calyx Extracts in 3T3-L1 Adipocytes

Hibiscus sabdariffa L. (sorrel) has been widely used in the development of tropical beverages and folk medicine. This study’s objective was to investigate the anti-obesity potential of sorrel calyx extracts (methanol and water) on 3T3-L1 adipocyte cells. Phytochemical content, antioxidant potential as DPPH (1, 1-Diphenyl-2-picrylhydrazyl) radical scavenging activity and ferric reducing antioxidant power (FRAP) and enzyme (α-glucosidase, α-amylase, and pancreatic lipase) inhibitory activities were determined in sorrel methanol extracts (SME) and sorrel water extracts (SWE). Effect of SWE and SME on lipid accumulation, lipolysis and apoptosis were tested in 3T3-L1 adipocyte differentiation and maintenance stage of cells at selected concentrations (200 - 1000 μg/ml) was studied. The total phenolic (GAE mg/100g dry weight) and total flavonoid (mg catechin equi/ 100g dry weight) contents in SME and SWE were 158.31 and 317.27 and 90.77 and 100.08. DPPH% inhibition (IC-50-mg/ml) and FRAP (mmol Fe [II]/100g dry weight) were 0.82 and 0.33 and 1799.13 and 2296.38 for SWE and SME, respectively. SME and SWE inhibited α-glucosidase, α-amylase, and pancreatic lipase activities by more than 40% at 4mg/ml. Significant (p < 0.05) reduction in lipid accumulation and increased glycerol release in 3T3-L1 cells was observed at concentrations ranged from 600 mg/ml of both extracts. Treating cells with SME-1000 μg/ml at differentiation resulted inhibition (p < 0.05) of lipid accumulation by 45% compared to untreated cells. Highest (p < 0.05) (35%) decrease in triglyceride content as well as higher glycerol release was seen in cells exposed to SME at the differentiation stage. Sorrel extracts induced apoptosis in adipocytes at higher concentrations with prominent effect of treating cells at differentiation stage. The results of this study showed effect of sorrel extracts in reduction of lipid accumulation and increase in lipolysis of 3T3-L1 cells.