Compound ES Research Articles

Inhibition of Osteoarthritis-Related Molecules by Isomucronulatol 7-O-β-d-glucoside and Ecliptasaponin A in IL-1β-Stimulated Chondrosarcoma Cell Model.

Osteoarthritis (OA) is the common form of arthritis and is characterized by disability and cartilage degradation. Although natural product extracts have been reported to have anti-osteoarthritic effects, the potential bioactivity of Ryupunghwan (RPH), a traditional Korean medicinal botanical formula that contains Astragalus membranaceus, Turnera diffusa, Achyranthes bidentata, Angelica gigas, Eclipta prostrata, Eucommia ulmoides, and Ilex paraguariensis, is not known well. Therefore, the inhibitory effects of single compounds isolated from RPH on the OA-related molecules were investigated using IL-1β-stimulated chondrosarcoma SW1353 (SW1353) cell model. Two bioactive compounds, isomucronulatol 7-O-β-d-glucoside (IMG) and ecliptasaponin A (ES) were isolated and purified from RPH using column chromatography, and then the structures were analyzed using ESI-MS, 1H-NMR, and 13C-NMR spectrum. The expression or amount of matrix metalloproteinase 13 (MMP13), COX1/2, TNF-α, IL-1β or p65 was determined by RT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA). RPH pretreatment reduced the expression and amounts of MMP13, and the expression of collagen II, COX1/2, TNF-α, IL-1β or p65, which were increased in IL-1β-stimulated SW1353 cells. IMG reduced the expression of all OA-related molecules, but the observed inhibitory effect was less than that of RPH extract. The other single compound ES showed the reduced expression of all OA-related molecules, and the effect was stronger than that in IMG (approximately 100 fold). Combination pretreatment of both single components remarkably reduced the expression of MMP13, compared to each single component. These synergic effects may provide potential molecular modes of action for the anti-osteoarthritic effects of RPH observed in clinical and animal studies.

How nature tunes isoenzyme activity in the multifunctional catalytic globin dehaloperoxidase from Amphitrite ornata

The coelomic hemoglobin of Amphitrite ornata, termed dehaloperoxidase (DHP), is the first known multifunctional catalytic globin to possess biologically-relevant peroxidase and peroxygenase activities. Although the two isoenzymes of DHP, A and B, differ in sequence by only 5 amino acids out of 137 residues, DHP B consistently exhibits a greater activity than isoenzyme A. To delineate the contributions of each amino acid substitution to the activity of either isoenzyme, the substitutions of the five amino acids were systematically investigated, individually and in combination, using 22 mutants. Biochemical assays and mechanistic studies demonstrated that the mutants that only contained the I9L substitution showed increased i) kcat values (peroxidase activity), ii) 5-Br-indole conversion and binding affinity (peroxygenase activity), and iii) rate of Compound ES formation (enzyme activation). Whereas the X-ray structures of the oxyferrous forms of DHP B (L9I) (1.96Å), DHP A (I9L) (1.20Å), and WT DHP B (1.81Å) showed no significant differences, UV–visible spectroscopy (ASoret/A380 ratio) revealed that the I9L substitution increased the 5-coordinate high-spin heme population characterized by the “open” conformation (i.e., distal histidine swung out of the pocket), which likely favors substrate binding. The positioning of the distal histidine closer to the heme cofactor in the solution state also appears to facilitate activation of DHP via the Compound ES intermediate. Taken together, the studies undertaken here shed light on the structure-function relationship in dehaloperoxidase, but also help to establish the foundation for understanding how enzymatic activity can be tuned in isoenzymes of a multifunctional catalytic globin.

Bis-Fe(IV): nature's sniper for long-range oxidation.

Iron-dependent enzymes are prevalent in nature and participate in a wide range of biological redox activities. Frequently, high-valence iron intermediates are involved in the catalytic events of iron-dependent enzymes, especially when the activation of peroxide or molecular oxygen is involved. Building on the fundamental framework of iron-oxygen chemistry, these reactive intermediates constantly attract significant attention from the enzymology community. During the past few decades, tremendous efforts from a number of laboratories have been dedicated to the capture and characterization of these intermediates to improve mechanistic understandings. In 2008, an unprecedented bis-Fe(IV) intermediate was reported in a c-type diheme enzyme, MauG, which is involved in the maturation of a tryptophan tryptophylquinone cofactor of methylamine dehydrogenase. This intermediate, although chemically equivalent to well-characterized high-valence iron intermediates, such as compound I, compound ES, and intermediate Q in methane monooxygenase, as well as the hypothetical Fe(V) species in Rieske non-heme oxygenases, is orders of magnitude more stable than these other high-valence species in the absence of its primary substrate. It has recently been discovered that the bis-Fe(IV) intermediate exhibits a unique near-IR absorption feature which has been attributed to a novel charge-resonance phenomenon. This review compares the properties of MauG with structurally related enzymes, summarizes the current knowledge of this new high-valence iron intermediate, including its chemical origin and structural basis, explores the formation and consequences of charge resonance, and recounts the long-range catalytic mechanism in which bis-Fe(IV) participates. Biological strategies for storing oxidizing equivalents with iron ions are also discussed.

Tyrosyl Radicals in Dehaloperoxidase: HOW NATURE DEALS WITH EVOLVING AN OXYGEN-BINDING GLOBIN TO A BIOLOGICALLY RELEVANT PEROXIDASE

Dehaloperoxidase (DHP) from Amphitrite ornata, having been shown to catalyze the hydrogen peroxide-dependent oxidation of trihalophenols to dihaloquinones, is the first oxygen binding globin that possesses a biologically relevant peroxidase activity. The catalytically competent species in DHP appears to be Compound ES, a reactive intermediate that contains both a ferryl heme and a tyrosyl radical. By simulating the EPR spectra of DHP activated by H2O2, Thompson et al. (Thompson, M. K., Franzen, S., Ghiladi, R. A., Reeder, B. J., and Svistunenko, D. A. (2010) J. Am. Chem. Soc. 132, 17501-17510) proposed that two different radicals, depending on the pH, are formed, one located on either Tyr-34 or Tyr-28 and the other on Tyr-38. To provide additional support for these simulation-based assignments and to deduce the role(s) that tyrosyl radicals play in DHP, stopped-flow UV-visible and rapid-freeze-quench EPR spectroscopic methods were employed to study radical formation in DHP when three tyrosine residues, Tyr-28, Tyr-34, and Tyr-38, were replaced either individually or in combination with phenylalanines. The results indicate that radicals form on all three tyrosines in DHP. Evidence for the formation of DHP Compound I in several tyrosine mutants was obtained. Variants that formed Compound I showed an increase in the catalytic rate for substrate oxidation but also an increase in heme bleaching, suggesting that the tyrosines are necessary for protecting the enzyme from oxidizing itself. This protective role of tyrosines is likely an evolutionary adaptation allowing DHP to avoid self-inflicted damage in the oxidative environment.

Kinetic Study of the Inhibition Mechanism of Dehaloperoxidase-Hemoglobin A by 4-Bromophenol

The mechanism of dehaloperoxidase-hemoglobin (DHP) inhibition by 4-bromophenol (4-BP) was investigated using Michealis-Menten and transient-state kinetic analyses. Transient-state kinetics using the stopped-flow technique to mix DHP and H2O2 in the presence of inhibitor concentrations less than 10-fold greater than the enzyme concentration show that 4-BP does not fully impede H2O2 entering the distal pocket to activate DHP. It is not clear whether an oxoferryl intermediate is formed under these conditions and there may be alternative pathways for H2O2 reaction in the 4-BP bound form of DHP. Two new species have been identified during the reaction of 4-BP bound form of DHP in the transient-state kinetic experiment by using Singular Value Decomposition (SVD) and global-fitting analysis. Rather than forming Compound ES in the unbound form, an inhibitor bound intermediate that possesses blue-shifted Soret band and a double peaked Q-band is observed. This intermediate is subsequently converted to the end-point species that is distinguished from Compound RH formed in the uninhibited enzyme. Bench-top mixing kinetics of DHP were conducted in order to determine the inhibitor binding constant and to understand the enzyme inhibition mechanism from a thermodynamic perspective. It was found that the inhibition constant, Ki, decreased from 2.56 mM to 0.15 mM over the temperature range from 283 to 298 K, which permits determination of the enthalpy and entropy for inhibitor binding as -135.5 ± 20.9 kJ/mol and 526.1 ± 71.9 J/(mol·K), respectively, leading to the conclusion that inhibitor binding is entropically driven.

Effect of distal His mutation on the peroxynitrite reactivity of Leishmania major peroxidase

The conserved distal histidine in peroxidases has been considered to play a major role as a general acid-base catalyst for heterolytic cleavage of an OO bond in H2O2. However, heme peroxidases react with peroxynitrite to form transient intermediates but the role of the distal histidine in this reaction is still unknown. In order to investigate catalytic roles of the histidine at the distal cavity, two Leishmania major peroxidase (LmP) mutants (H68E, H68V) were prepared. The rate of transition from ferric H68V to Compound ES by H2O2 is decreased by approximately five orders of magnitude relative to wild type, which is consistent with electron donor oxidation data where the H68V is ~1000 fold less active than wild type. In the reaction with peroxynitrite, the formation rate of intermediates in the mutants is not significantly lower than that for the wild type, indicating that the His68 has no major role in homolytic cleavage of an OO bond in peroxynitrite. EPR spectroscopic data suggest that the transient intermediates formed by the reaction of LmP with H2O2 exhibits an intense and stable signal similar to CCP Compound ES whereas in case of the reaction with peroxynitrite, this signal disappears, indicating that the transient intermediate is Compound II. Rapid kinetics data suggest that the distal His68 mutants display higher decay rates of Compound II than wild type. Thus, His68 mutations minimize Compound II formation (inactive species in peroxynitrite scavenging cycles) by increasing decay rates during the steady state and results in higher peroxynitrite degrading activity.

The Role of the Distal Histidine in H2O2 Activation and Heme Protection in both Peroxidase and Globin Functions

The distal histidine mutations of dehaloperoxidase-hemoglobin A (DHP A) to aspartate (H55D) and asparagine (H55N) have been prepared to study the role played by the distal histidine in both activation and protection against oxidation by radicals in heme proteins. The H55D and H55N mutants of DHP A have ~6-fold and ~11-fold lower peroxidase activities than wild type enzyme toward the oxidation of 2,4,6-trichlorophenol (TCP) to yield 2,6-dichloroquinone (DCQ) in the presence of H(2)O(2). The origin of the lower rate constants may be the solvent-exposed conformations of distal D55 and N55, which would have the dual effect of destabilizing the binding of H(2)O(2) to the heme iron, and of removing the acid-base catalyst necessary for the heterolytic O-O bond cleavage of heme-bound H(2)O(2) (i.e., compound 0). The partial peroxidase activity of H55D can be explained if one considers that there are two conformations of the distal aspartate (open and closed) by analogy with the distal histidine. We hypothesize that the distal aspartate has an active conformation in the distal pocket (closed). Although the open form is observed in the low-temperature X-ray crystal structure of ferric H55D, the closed form is observed in the FTIR spectrum of the carbonmonoxy form of the H55D mutant. Consistent with this model, the H55D mutant also shows inhibition of TCP oxidation by 4-bromophenol (4-BP). Consistent with the protection hypothesis, compound ES, the tyrosyl radical-containing ferryl intermediate observed in WT DHP A, was not observed in H55D.

Decay of Compound ES in Dehaloperoxidase-Hemoglobin

Dehaloperoxidase (DHP) is a respiratory hemoglobin (Hb) that catalyzes the conversion of trihalophenols to dihaloquinones in the presence of hydrogen peroxide. Ferric heme states of the resting DHP and the free radical intermediates formed under H2O2 treatment were studied by low temperature EPR spectroscopy in the range of reaction time of 50 ms - 2 min at three different pH values. Two high spin ferric heme forms were identified in the resting enzyme and assigned to the open and closed conformations of the distal histidine, His55. Two free radicals were found in DHP activated by H2O2: the radical associated with Compound ES has been assigned to Tyr34, the other radical - to Tyr38. The Tyr34 radical is formed with a very high relative yield (almost 100% of heme), atypical of other globins. The HPLC analysis of the reaction products showed a pH dependent formation of covalent heme-to-protein cross-links. The stable DHP Compound RH formed under H2O2 in the absence of substrates is proposed to be a state with the ferric heme covalently cross-linked to Tyr34. A kinetic model of the experimental data suggests that formation of Compound RH or the Tyr38 radical are two alternative routes of Compound ES decay. Which route is taken depends on the conformation of His55: in the less populated closed conformation, the Tyr38 radical is formed, but in the major open conformation, Compound ES decays yielding Compound RH, a product of safe termination of the two oxidizing equivalents of H2O2 when no substrate is available.

Effect of H55D Mutation on Kinetics and Structure of Dehaloperoxidase-Hemoglobin A

The H55D mutant of dehaloperoxidase-hemoglobin A (DHP A) has been prepared to mimic the H64D mutant of Sperm Whale myoglobin (SWMb), which has the highest peroxidase activity of any myoglobin mutant studied to date. The high peroxidase activity has been attributed to the rapid formation as well as the prolonged lifetime of compound I following addition of H2O2. However, unlike H64D SWMb, the H55D mutant of DHP A has ∼13-fold lower peroxidase activity towards oxidation of 2,4,6-trichlorophenol (TCP) into 2,6-dichloroquinone (DCQ) in the presence of H2O2. The origin of the lower rate constant may be the solvent-exposed conformation of D55, which has the effect of removing the acid-base catalyst necessary for heterolytic cleavage O-O bond in Fe-bound H2O2. These observations further support the hypothesis that the flexibility of distal histidine in wild-type DHP A, plays a crucial role in enzyme function. Remarkably, the H55D mutant also shows inhibition by 4-bromophenol (4-BP) in the oxidation of TCP, which is substantiated by the X-ray crystal structure of 4-BP bound to the internal inhibitor binding site observed in wild type DHP A and B. The inhibitor complex is an unusual internally bound molecule, completely surrounded by the protein. Compound ES, a tyrosine radical species associated with the oxoferryl intermediate detected in DHP A, is not observed in H55D. Thus, the H55D mutant is a key test case for the role of electron transfer in the DHP A mechanism.

Site(s) of Tyrosyl Radical Formation in Dehaloperoxidase A and B

Dehaloperoxidase (DHP) is a dual-function hemoprotein that, in addition to being the putative coelomic hemoglobin of Amphitrite ornata, exhibits a broad substrate specificity for the catalytic oxidation of mono-, di-, and trisubstituted halophenols, thus distinguishing DHP as the first globin shown to possess a biologically relevant peroxidase activity. Both isoenzymes of DHP, termed A and B, have been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that utilizes hydrogen peroxide as the oxidant. We have shown that the initially formed heme intermediate in wild-type DHP is not Compound I as is often the case in peroxidases, but rather is a combination of an iron(IV)-oxo and a tyrosyl radical that together have similarity to the Compound ES intermediate of cytochrome c peroxidase. In order to possibly identify the site(s) of this radical species in DHP, we have studied the tyrosine mutants DHP A (Y34F), DHP A (Y38F), DHP A (Y34F/Y38F), DHP B (Y28F), DHP B (Y38F), and DHP B (Y28F/Y38F), and studied their reaction with hydrogen peroxide using a combination of stopped-flow UV-visible and rapid-freeze quench electron paramagnetic resonance spectroscopies. Spectroscopic evidence for the formation of both Compound I and Compound ES will be presented. We have further characterized these mutants using biochemical assays to determine their effect on the catalytic activity of the enzyme, and relate these results to the structure of the heme active site and the formation of the catalytically-attenuated species Compound RH. Such mutagenesis studies of DHP provide critical insight into the mechanistic details of the H2O2-dependent oxidative dehalogenation reaction catalyzed by dehaloperoxidase, present a clearer description of the function of DHP at the molecular level, and lead to a better understanding of the paradigms of globin structure-function relationships.

Compound ES of Dehaloperoxidase Decays via Two Alternative Pathways Depending on the Conformation of the Distal Histidine

Dehaloperoxidase (DHP) is a respiratory hemoglobin (Hb) that has been shown to catalyze the conversion of trihalophenols to dihaloquinones in the presence of hydrogen peroxide. Ferric heme states of the resting DHP and the free radical intermediates formed under H2O2 treatment were studied by low-temperature electron paramagnetic resonance spectroscopy in the range of reaction times from 50 ms to 2 min at three different pH values. Two high-spin ferric heme forms were identified in the resting enzyme and assigned to the open and closed conformations of the distal histidine, His55. Two free radicals were found in DHP activated by H2O2: the radical associated with Compound ES (the enzyme with the heme in the oxoferryl state and a radical on the polypeptide chain) has been assigned to Tyr34, and the other radical has been assigned to Tyr38. The Tyr34 radical is formed with a very high relative yield (almost 100% of heme), atypical of other globins. High-performance liquid chromatography analysis of the reaction products showed a pH-dependent formation of covalent heme-to-protein cross-links. The stable DHP Compound RH, formed under H2O2 in the absence of the trihalophenol substrates, is proposed to be a state with the ferric heme covalently cross-linked to Tyr34. A kinetic model of the experimental data suggests that formation of Compound RH and formation of the Tyr38 radical are two alternative routes of Compound ES decay. Which route is taken depends on the conformation of His55: in the less populated closed conformation, the Tyr38 radical is formed, but in the major open conformation, Compound ES decays, yielding Compound RH, a product of safe termination of the two oxidizing equivalents of H2O2 when no substrate is available.

Reaction of N-hydroxyguanidine with the ferrous–oxy state of a heme peroxidase cavity mutant: A model for the reactions of nitric oxide synthase

Yeast cytochrome c peroxidase was used to construct a model for the reactions catalyzed by the second cycle of nitric oxide synthase. The R48A/W191F mutant introduced a binding site for N-hydroxyguanidine near the distal heme face and removed the redox active Trp-191 radical site. Both the R48A and R48A/W191F mutants catalyzed the H 2O 2 dependent conversion of N-hydroxyguanidine to N-nitrosoguanidine. It is proposed that these reactions proceed by direct one-electron oxidation of NHG by the Fe +4 O center of either Compound I (Fe +4 O, porph + ) or Compound ES (Fe +4 O, Trp + ). R48A/W191F formed a Fe +2O 2 complex upon photolysis of Fe +2CO in the presence of O 2, and N-hydroxyguanidine was observed to react with this species to produce products, distinct from N-nitrosoguanidine, that gave a positive Griess reaction for nitrate + nitrite, a positive Berthelot reaction for urea, and no evidence for formation of NO . It is proposed that HNO and urea are produced in analogy with reactions of nitric oxide synthase in the pterin-free state.

Characterization of the Radical Intermediates of Dehaloperoxidase A and B from Amphitrite Ornata

The enzyme intermediates of dehaloperoxidase (DHP) from the marine worm Amphitrite ornata are unique within both the globin and cytochrome c peroxidase superfamilies. Both isoenzymes of DHP, termed A and B, have been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that utilizes hydrogen peroxide as a co-substrate. We have shown that the initially formed heme intermediate in this reaction is not Compound I as is often the case in peroxidases, but rather is a combination of an iron(IV)-oxo (Compound II) and a tyrosyl radical that together have similarity to the Compound ES intermediate of cytochrome c peroxidase. In order to possibly identify the origin of this radical species in DHP, we have expressed the tyrosine mutants DHP A (Y34F), DHP A (Y38F), DHP A (Y34F/Y38F), and DHP B (Y38F), and studied their reaction with hydrogen peroxide using a combination of stopped-flow UV-visible and rapid-freeze quench electron paramagnetic resonance spectroscopies. Although each mutant exhibited an average signal at g ≈ 2.0058 confirming the presence of a protein radical, significant differences in the lineshape and width of each radical was observed. We have further characterized these mutants using biochemical assays to determine their effect on the catalytic activity of the enzyme, and relate these results to the structure of the heme active site. Such mutagenesis studies of DHP provide critical insight into the mechanistic details of the H2O2-dependent oxidative dehalogenation reaction catalyzed by dehaloperoxidase, present a clearer description of the function of DHP at the molecular level, and lead to a better understanding of the paradigms of globin structure-function relationships.

The Mechanism of Oxidative Halophenol Dehalogenation by Amphitrite ornata Dehaloperoxidase Is Initiated by H2O2 Binding and Involves Two Consecutive One-Electron Steps: Role of Ferryl Intermediates

The enzymatic globin, dehaloperoxidase (DHP), from the terebellid polychaete Amphitrite ornata is designed to catalyze the oxidative dehalogenation of halophenol substrates. In this study, the ability of DHP to catalyze this reaction by a mechanism involving two consecutive one-electron steps via the normal order of addition of the oxidant cosubstrate (H(2)O(2)) before organic substrate [2,4,6-trichlorophenol (TCP)] is demonstrated. Specifically, 1 equiv of H(2)O(2) will fully convert 1 equiv of TCP to 2,6-dichloro-1,4-benzoquinone, implicating the role of multiple ferryl [Fe(IV)O] species. A significant amount of heterolytic cleavage of the O-O bond of cumene hydroperoxide, consistent with transient formation of a Compound I [Fe(IV)O/porphyrin pi-cation radical] species, is observed upon its reaction with ferric DHP. In addition, a more stable high-valent Fe(IV)O-containing DHP intermediate [Compound II (Cpd II) or Compound ES] is characterized by UV-visible absorption and magnetic circular dichroism spectroscopy. Spectral similarities are seen between this intermediate and horse heart myoglobin Cpd II. It is also shown in single-turnover experiments that the DHP Fe(IV)O intermediate is an active oxidant in halophenol oxidative dehalogenation. Furthermore, reaction of DHP with 4-chlorophenol leads to a dimeric product. The results presented herein are consistent with a normal peroxidase order of addition of the oxidant cosubstrate (H(2)O(2)) followed by organic substrate (TCP) and indicate that the enzymatic mechanism of DHP-catalyzed oxidative halophenol dehalogenation involves two consecutive one-electron steps with a dissociable radical intermediate.

Spectroscopic Probes of the Reactive Intermediates of Dehaloperoxidase from Amphitrite ornata

The enzyme intermediates of dehaloperoxidase (DHP) from the marine worm Amphitrite ornata are unique within both the globin and cytochrome c peroxidase superfamilies. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. We show that the initially formed heme intermediate in this reaction is not Compound I as is often the case in peroxidases, but rather is a combination of Compound II and a tyrosyl radical that has similarity to the Compound ES intermediate of cytochrome c peroxidase. Using stopped-flow UV-visible spectroscopy, we provide a detailed kinetic description for the reaction of pre-formed DHP Compound II and tyrosyl radical with the substrate 2,4,6-trichlorophenol, and demonstrate the catalytic competency of this intermediate in generating the product 2,4-dichloroquinone. Furthermore, using rapid-freeze-quench electron paramagnetic resonance spectroscopy, we detected a signal at g ≈ 2.0058, confirming the presence of a protein radical in DHP Compound II, and assign it as a tyrosyl radical based upon mutagenesis studies and structural arguments. In the absence of a halophenol substrate, the DHP Compound II + tyrosyl radical intermediate decomposes to a new and significantly less active species, termed Compound RH, which is unique to dehaloperoxidase. We propose that this intermediate plays a protective role against heme bleaching. While unreactive toward further oxidation, Compound RH can be reduced and subsequently bind dioxygen, generating oxyferrous DHP, which may represent the catalytic link between the peroxidase and oxygen-transport activities in this bifunctional protein.

Characterization of Dehaloperoxidase Compound ES and Its Reactivity with Trihalophenols

Dehaloperoxidase (DHP), the oxygen transport hemoglobin from the terebellid polychaete Amphitrite ornata, is the first globin identified to possess a biologically relevant peroxidase activity. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. Herein, we demonstrate that the first detectable intermediate following the addition of hydrogen peroxide to ferric DHP contains both a ferryl heme and a tyrosyl radical, analogous to Compound ES of cytochrome c peroxidase. Furthermore, we provide a detailed kinetic description for the reaction of preformed DHP Compound ES with the substrate 2,4,6-trichlorophenol and demonstrate the catalytic competency of this intermediate in generating the product 2,4-dichloroquinone. Using rapid-freeze-quench electron paramagnetic resonance spectroscopy, we detected a g approximately 2.0058 signal confirming the presence of a protein radical in DHP Compound ES. In the absence of substrate, DHP Compound ES evolves to a new species, Compound RH, which is functionally unique to dehaloperoxidase. We propose that this intermediate plays a protective role against heme bleaching. While unreactive toward further oxidation, Compound RH can be reduced and subsequently bind dioxygen, generating oxyferrous DHP, which may represent the catalytic link between peroxidase and oxygen transport activities in this bifunctional protein.

Characterization of the Peroxidase Mechanism upon Reaction of Prostacyclin Synthase with Peracetic Acid. Identification of a Tyrosyl Radical Intermediate

Prostacyclin synthase (PGIS) is a membrane-bound class III cytochrome P450 that catalyzes an isomerization of prostaglandin H(2), an endoperoxide, to prostacyclin. We report here the characterization of the PGIS intermediates in reactions with other peroxides, peracetic acid (PA), and iodosylbenzene. Rapid-scan stopped-flow experiments revealed an intermediate with an absorption spectrum similar to that of compound ES (Cpd ES), which is an oxo-ferryl (Fe(IV)O) plus a protein-derived radical. Cpd ES, formed upon reaction with PA, has an X-band (9 GHz) EPR signal of g = 2.0047 and a half-saturation power, P(1/2), of 0.73 mW. High-field (130 GHz) EPR reveals the presence of two species of tyrosyl radicals in Cpd ES with their g-tensor components (g(x), g(y), g(z)) of 2.00970, 2.00433, 2.00211 and 2.00700, 2.00433, 2.00211 at a 1:2 ratio, indicating that one is involved in hydrogen bonding and the other is not. The line width of the g = 2 signal becomes narrower, while its P(1/2) value becomes smaller as the reaction proceeds, indicating migration of the unpaired electron to an alternative site. The rate of electron migration ( approximately 0.2 s(-1)) is similar to that of heme bleaching, suggesting the migration is associated with the enzymatic inactivation. Moreover, a g = 6 signal that is presumably a high-spin ferric species emerges after the appearance of the amino acid radical and subsequently decays at a rate comparable to that of enzymatic inactivation. This loss of the g = 6 species thus likely indicates another pathway leading to enzymatic inactivation. The inactivation, however, was prevented by the exogenous reductant guaiacol. The studies of PGIS with PA described herein provide a mechanistic model of a peroxidase reaction catalyzed by the class III cytochromes P450.

EPR and ENDOR Studies of Cryoreduced Compounds II of Peroxidases and Myoglobin. Proton-Coupled Electron Transfer and Protonation Status of Ferryl Hemes

The nature of the [Fe(IV)-O] center in hemoprotein Compounds II has recently received considerable attention, as several experimental and theoretical investigations have suggested that this group is not necessarily the traditionally assumed ferryl ion, [Fe(IV)=O]2+, but can be the protonated ferryl, [Fe(IV)-OH]3+. We show here that cryoreduction of the EPR-silent Compound II by gamma-irradiation at 77 K produces Fe(III) species retaining the structure of the precursor [Fe(IV)=O]2+ or [Fe(IV)-OH]3+, and that the properties of the cryogenerated species provide a report on structural features and the protonation state of the parent Compound II when studied by EPR and 1H and 14N ENDOR spectroscopies. To give the broadest view of the properties of Compounds II we have carried out such measurements on cryoreduced Compounds II of HRP, Mb, DHP and CPO and on CCP Compound ES. EPR and ENDOR spectra of cryoreduced HRP II, CPO II and CCP ES are characteristic of low-spin hydroxy-Fe(III) heme species. In contrast, cryoreduced "globins", Mb II, Hb II, and DHP II, show EPR spectra having lower rhombicity. In addition the cryogenerated ferric "globin" species display strongly coupled exchangeable (1)H ENDOR signals, with A max approximately 20 MHz and a iso approximately 14 MHz, both substantially greater than for hydroxide/water ligand protons. Upon annealing at T > 180 K the cryoreduced globin compounds II relax to the low-spin hydroxy-ferric form with a solvent kinetic isotope effect, KIE > 6. The results presented here together with published resonance Raman and Mossbauer data suggest that the high-valent iron center of globin and HRP compounds II, as well as of CCP ES, is [Fe(IV)=O]2+, and that its cryoreduction produces [Fe(III)-O]+. Instead, as proposed by Green and co-workers, CPO II contains [Fe(IV)-OH]3+ which forms [Fe(III)-OH]2+ upon radiolysis. The [Fe(III)-O]+ generated by cryoreduction of HRP II and CCP ES protonate at 77 K, presumably because the heme is linked to a distal-pocket hydrogen bonding/proton-delivery network through an H-bond to the "oxide" ligand. The data also indicate that Mb and HRP compounds II exist as two major conformational substates.

The marine sphingolipid-derived compound ES 285 triggers an atypical cell death pathway

The isolation of new molecules from marine sources opens the door to their possible therapeutic use against tumors and other pathological conditions. Indeed, we recently defined the cytotoxicity of ES 285, obtained from the clam Mactromeris polynima, and its affects on the cells microfilament but not the microtubule network. Considering the analogy between ES 285 and sphingosine-related lipids, we wondered whether ES 285 might affect the activity of PKC at the intracellular level. While we anticipated that ES 285 might inhibit PKC, it turns out that in contrast it serves to activate PKC at the cellular level. Indeed, like other sphingosine-related lipids, ES 285 induces the phosphorylation of MARCKS. Additionally, we further examined the cytotoxicity of ES 285 to elucidate the molecular mechanisms through which this compound triggers apoptosis. When the influence of ES 285 on "cell death markers" was assessed, it became clear that ES285 activates caspase 3 and 12, and that it modified the phosphorylation of p53. In contrast, ES 285 does not affect other pathways widely implicated in regulating cell survival/apoptosis, such as JNK, Erks or Akt. Thus, these data suggest that ES 285-triggers an atypical cell death program when compared to other sphingosine-dependent apoptosis pathways.

Rapid kinetics investigations of peracid oxidation of ferric cytochrome P450cam: Nature and possible function of compound ES

Previously, we reported spectroscopic properties of cytochrome P450cam compound I, (ferryl iron plus a porphyrin π-cation radical (Fe IV = O/Por + )), as well as compound ES (Fe IV = O/Tyr ) in reactions of substrate-free ferric enzyme with m-chloroperbenzoic acid [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300-9]. Compound ES arises by intramolecular electron transfer from nearby tyrosines to the porphyrin π-cation radical of Compound I, and has been characterized by rapid-freeze-quench-Mössbauer/EPR spectroscopy; the tyrosyl radical was assigned to Tyr96 for wild type or to Tyr75 for the Tyr96Phe variant [V. Schünemann, F. Lendzian, C. Jung, J. Contzen, A.L. Barra, S.G. Sligar, A.X. Trautwein, J. Biol. Chem. 279 (2004) 10919–10930]. Here we report rapid-scanning stopped-flow studies of the reactions of peracids with substrate-free ferric Y75F, Y96F, and Y96F/Y75F P450cam variants, showing how these active site changes influence electron transfer from nearby tyrosines and affect formation of intermediates. Curiously, rates of generation of Compounds I and ES for both single mutants were not very different from wild type. Contrasting with the earlier EPR results, the Y96F/Y75F variant was also shown to form an ES-like species, but more slowly. When substrate is not present, or is improperly bound, compound I rapidly converts to compound ES, which can be reduced to form H 2O and ferric P450, thus avoiding the modification of nearby protein groups or release of reactive oxygen species.