Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury

There has been considerable controversy regarding the role of oxygen free radicals as important mediators of cell damage in reperfused myocardium. This controversy regards whether superoxide and hydroxyl free radicals are generated on reperfusion and if these radicals actually cause impaired contractile function. In this study, EPR studies using the spin trap 5,5-dimethyl-1-pyroline-n-oxide (DMPO) demonstrate the formation of .OH and R. free radicals in the reperfused heart. EPR signals of DMPO-OH, aN = aH = 14.9 G, and DMPO-R aN = 15.8 G aH = 22.8 G are observed, with peak concentrations during the first minute of reperfusion. It is demonstrated that these radicals are derived from .O2- since reperfusion in the presence of enzymatically active recombinant human superoxide dismutase markedly reduced the formation of these signals while inactive recombinant human superoxide dismutase had no effect. On reperfusion with perfusate pretreated to remove adventitial iron, the concentration of the DMPO-OH signal was increased 2-fold and a 4-fold decrease in the DMPO-R signal was observed demonstrating that iron-mediated Fenton chemistry occurs. Hearts reperfused with recombinant human superoxide dismutase exhibited improved contractile function in parallel with the marked reduction in measured free radicals. In order to determine if the reperfusion free radical burst results in impaired contractile function, simultaneous measurements of free radical generation and contractile function were performed. A direct relationship between free radical generation and subsequent impaired contractile function was observed. These studies suggest that superoxide derived .OH and R. free radicals are generated in the reperfused heart via Fenton chemistry. These radicals appear to be key mediators of myocardial reperfusion injury.     

An organic radical in the lysine 2,3-aminomutase reaction.

Lysine 2,3-aminomutase from Clostridium SB4 has been studied by electron paramagnetic resonance (EPR) spectroscopy at 77 K. Although the reaction catalyzed by this enzyme is similar to rearrangements catalyzed by enzymes requiring adenosylcobalamin, lysine 2,3-aminomutase does not utilize this cofactor. The enzyme instead contains iron-sulfur clusters, cobalt, and pyridoxal phosphate and is activated by S-adenosylmethionine. Subsequent to a reductive incubation procedure that is required to activate the enzyme, EPR studies reveal the appearance of an organic radical signal (g = 2.001) upon addition of both L-lysine and S-adenosylmethionine. The radical signal is complex, having multiple hyperfine transitions. The total radical concentration is proportional to enzyme activity and decreases in parallel with the approach to chemical equilibrium between alpha-lysine and beta-lysine. The signal changes over the time course of the reaction in a way that suggests the presence of more than one radical species, with different relative proportions of species in the steady state and equilibrium state. Isotopic substitution experiments show that unpaired spin density resides on the molecular framework of lysine and that solvent-exchangeable protons do not participate in strong hyperfine coupling to the radical. The results indicate that lysine radicals participate in the rearrangement mechanism.     

Structure of a substrate radical intermediate in the reaction of lysine 2,3-aminomutase.

Electron paramagnetic resonance (EPR) spectroscopy has been used to characterize an organic radical that appears in the steady state of the reaction catalyzed by lysine 2,3-aminomutase from Clostridium SB4. Results of a previous electron paramagnetic resonance (EPR) study [Ballinger, M. D., Reed, G. H., & Frey, P. A. (1992) Biochemistry 31, 949-953] demonstrated the presence of EPR signals from an organic radical in reaction mixtures of the enzyme. The materialization of these signals depended upon the presence of the enzyme, all of its cofactors, and the substrate, lysine. Changes in the EPR spectrum in response to deuteration in the substrate implicated the carbon skeleton of lysine as host for the radical center. This radical has been further characterized by EPR measurements on samples with isotopically substituted forms of lysine and by analysis of the hyperfine splittings in resolution-enhanced spectra by computer simulations. Changes in the hyperfine splitting patterns in EPR spectra from samples with [2-2H]lysine and [2-13C]-lysine show that the paramagnetic species is a pi-radical with the unpaired spin localized primarily in a p orbital on C2 of beta-lysine. In the EPR spectrum of this radical, the alpha-proton, the beta-nitrogen, and the beta-proton are responsible for the hyperfine structure. Analysis of spectra for reactions initiated with L-lysine, [3,3,4,4,5,5,6,6-2H8]lysine, [2-2H]lysine, perdeuteriolysine, [alpha-15N]lysine, and [alpha-15N,2-2H]lysine permit a self-consistent assignment of hyperfine splittings.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct FeS cluster involvement in generation of a radical in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (KAM) belongs to a class of enzymes that use FeS clusters and S-adenosyl-L-methionine to initiate radical-dependent chemistry. Selenium K-edge X-ray absorption spectroscopic analysis of KAM poised at various stages of catalysis, in the presence of selenomethionine or Se-adenosyl-L-selenomethionine, reveals that the cofactor is cleaved only in the presence of dithionite and the substrate analogue trans-4,5-dehydrolysine. A new Fourier transform peak at 2.7 A, assigned as a Se-Fe interaction, appears concomitant with this cleavage. This is the first demonstration of a direct interaction of S-adenosyl-L-methionine, or its cleavage products, with the FeS cluster in this class of enzymes.     

The enzymatic oxidation of Desferal to a nitroxide free radical

Desferrioxamine mesylate (Desferal), a transition metal ion chelator, has been used to inhibit the in vitro redox cycling of transition metal ions. ESR spectroscopy was utilized to detect and identify Desferal's one-electron oxidation product. We demonstrate that a horseradish peroxidase/H₂O₂ system, a xanthine oxidase/hypoxanthine system, and a hydroxyl radical-generating system are all capable of oxidizing Desferal to a nitroxide free radical. The same 9-line ESR spectrum (g = 2.0065, a^N = 7.85 G, a^H(2) = 6.35 G) was detected in all of the above systems. We, therefore, stress that care must be taken when using Desferal as a transition metal ion chelator to keep its concentration low enough to minimize these reactions, or to use a different metal ion chelator.     

Amorpha-4,11-diene synthase: mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate

Recombinant amorpha-4,11-diene synthase from Artemisia annua, expressed in Escherichia coli, was incubated with the deuterium-labeled farnesyl diphosphates, (1R)-[1-(2)H]FPP, (1S)-[1-(2)H]FPP, and [1,1-(2)H2]FPP. GC-MS analysis of amorpha-4,11-diene formed from the deuterated FPPs shows that the deuterium atoms are retained in the product. Furthermore, analysis of the MS-spectra obtained with the differently labeled substrate indicates that the H-1si-proton of FPP is transferred during the cyclization reaction to carbon 10 of amorphadiene while the H-1re-proton of FPP is retained on C-6 of the product. Proton NMR and COSY experiments proved that the original H-1si-proton of FPP is located at C-10 of amorpha-4,11-diene as a result of a 1,3-hydride shift following initial 1,6-ring closure. The results obtained support the previously suggested mechanism for the cyclization of farnesyl diphosphate by amorph-4,11-diene synthase involving isomerization of FPP to (R)-nerolidyl diphosphate (NPP), ionization of NPP, and C-1,C-6-ring closure to generate a bisabolyl cation, followed by a 1,3-hydride shift, 1,10-ring closure to generate the amorphane skeleton, and deprotonation at either C-12 or C-13 to afford the final product (1S,6R,7R,10R)-amorpha-4,11-diene.     

Phenylpropanoid glycoside analogues: enzymatic synthesis, antioxidant activity and theoretical study of their free radical scavenger mechanism

Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols.     

Cytochrome P450: substrate and prosthetic-group free radicals generated during the enzymatic cycle

During the enzymatic cycle of the cytochromes P450, dioxygen binds to the ferrous haemprotein when the resting ferric haemprotein has undergone a one-electron oxidation after substrate binding. A further one-electron reduction generates an intermediate that is isoelectronic with a peroxide dianion coordinated to a ferric iron. Heterolytic cleavage of the omicron--omicron bond generates water and a species which is formally an oxene (oxygen atom) coordinated by iron(III). However, on the basis of model reactions and by analogy to the catalases and peroxidases, this active oxidizing intermediate is formulated as an oxo-FeIV porphyrin pi-cation radical. The radical is stabilized by delocalization on the porphyrin macrocycle and the high oxidation state is achieved by oxidizing both the metal and the porphyrin ring of the haemprotein. Hydrogen atom abstraction from a saturated hydrocarbon substrate generates a substrate free radical, constrained by the protein binding site, and the equivalent of a hydroxyl radical bound to iron(III). Coupling of the 'hydroxy' and substrate radicals generates hydroxylated product and resting protein. For olefins an initial electron transfer to oxidized haemprotein gives a substrate cation radical. Further reaction of this radical can give the epoxide, the principal product; an aldehyde or ketone by rearrangement; or an alkylated haemprotein resulting in suicide inhibition.     

The enzymatic reduction of actinomycin D to a free radical species

Actinomycin D is an antitumor antibiotic in current clinical use. The ability of this and other antitumor antibiotics to undergo a reductive metabolism to produce free radical species has raised considerable interest in the literature in the past few years. The ability of actinomycin D to undergo a reductive metabolism was investigated using a ferredoxin reductase/NADPH system. This enzyme system has been used by a number of authors as a model for an enzymatic drug reducing system. In this study radical production was measured using direct ESR spectroscopy, the spin trapping technique, and oxygen consumption. It was shown that under anaerobic conditions the ferredoxin reductase/NADPH system could reduce actinomycin D to produce a semiquinone-imine free radical (aN = 2.8 (2N); aH = 2.8 (3H)). This radical production was found to be both drug and NADPH dependent. The effect of DNA on the drug's metabolism was also investigated. This was thought to be important because the proposed therapeutic action of the drug is centered on the DNA. Addition of calf thymus DNA to the reaction system abolished the signal produced by the actinomycin D, suggesting that intercalated actinomycin D is not a suitable substrate for ferredoxin reductase. Under aerobic conditions the ferredoxin reductase/NADPH/actinomycin D system generated the superoxide anion radical by reducing molecular oxygen. Evidence for this was obtained by spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The DMPO-superoxide radical adduct was produced (aN = 14.4 G; aH beta = 11.4 G; aH gamma = 1.3 G). Production of this adduct was drug and NADPH dependent, and was inhibited by superoxide dismutase. Superoxide production was also monitored by oxygen consumption studies.     

Binding energy in the one-electron reductive cleavage of S-adenosylmethionine in lysine 2,3-aminomutase, a radical SAM enzyme

The common step in the actions of members of the radical SAM superfamily of enzymes is the one-electron reductive cleavage of S-adenosyl-l-methionine (SAM) into methionine and the 5'-deoxyadenosyl radical. The source of the electron is the [4Fe-4S]1+ cluster characterizing the radical SAM superfamily, to which SAM is directly ligated through its methionyl carboxylate and amino groups. The energetics of the reductive cleavage of SAM is an outstanding question in the actions of radical SAM enzymes. The energetics is here reported for the action of lysine 2,3-aminomutase (LAM), which catalyzes the interconversion of l-lysine and l-beta-lysine. From earlier work, the reduction potential of the [4Fe-4S]2+/1+ cluster in LAM is -0.43 V with SAM bound to the cluster (Hinckley, G. T., and Frey, P. A. (2006) Biochemistry 45, 3219-3225), 1.4 V higher than the reported value for trialkylsulfonium ions in solution. The midpoint reduction potential upon binding l-lysine has been estimated to be -0.6 V from the values of midpoint potentials measured with SAM bound to the cluster and l-alanine in place of l-lysine, with S-adenosyl-l-homocysteine (SAH) bound to the cluster in the presence of l-lysine, and with SAH bound to the cluster in the presence of l-alanine or of l-alanine and ethylamine in place of l-lysine. The reduction potential for SAM has been estimated to be -0.99 V from the measured value for S-3',4'-anhydroadenosyl-l-methionine. The reduction potential for the [4Fe-4S] cluster is lowered 0.17 V by the binding of lysine to LAM, and the binding of SAM to the [4Fe-4S] cluster in LAM elevates its reduction potential by 0.81 V. Thus, the binding of l-lysine to LAM contributes 4 kcal mol-1, and the binding of SAM to the [4Fe-4S] cluster in LAM contributes 19 kcal mol-1 toward lowering the barrier for reductive cleavage of SAM from 32 kcal mol-1 in solution to 9 kcal mol-1 at the active site of LAM.     

Even free radicals should follow some rules: A Guide to free radical research terminology and methodology

Free radicals and oxidants are now implicated in physiological responses and in several diseases. Given the wide range of expertise of free radical researchers, application of the greater understanding of chemistry has not been uniformly applied to biological studies. We suggest that some widely used methodologies and terminologies hamper progress and need to be addressed. We make the case for abandonment and judicious use of several methods and terms and suggest practical and viable alternatives. These changes are suggested in four areas: use of fluorescent dyes to identify and quantify reactive species, methods for measurement of lipid peroxidation in complex biological systems, claims of antioxidants as radical scavengers, and use of the terms for reactive species.     

Light-induced free radical alkylation of polynucleotides and their enzymatic digestion.

Ultraviolet light-induced free radical alkylation with 2-propanol or D-ribose, initiated with di-tert-butyl peroxide, of poly (G), poly (U20G), and poly(A) led to the substitution of the appropriate group for the H-8 atom of the purines and addition across the 5,6-double bond of the pyrimidines. The alkylated polynucleotides were subjected to nucleolytic digestion with several nucleases. T1-RNase digestion of poly(G) irradiated with 2-propanol gave a mixture of the modified and non-modified mononucleotides. Similarly, pancreatic RNase digestion of the irradiated poly(U20G) resulted in a mixture of the appropriate mononucleotides. A T2-RNase treatment of poly(A) irradiated with 2-propanol gave the modified Ado-21:3'-P, while T2-RNase digestion of poly(A) irradiated with D-ribose led to the cyclic modified mononucleotides, in addition to the modified mononucleotides.     

Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis

High levels of NaCN (20 to 250 mM) were required to inhibit cyclooxygenase catalysis and cause extended lag periods (up to 1.6 min), whereas CO failed to inhibit catalysis. This NaCN inhibition was easily overcome by endogenous or exogenous hydroperoxides. Added hydroperoxides acted to eliminate lag periods without undergoing net conversion to other chemical species. In addition, experiments with glutathione peroxidase inhibition showed that hydroperoxides were essential not only in the early phases, but throughout catalysis. In spectrophotometric experiments, NaCN formed a complex with ferriheme cyclooxygenase (Kd = 1.3 mM) and inhibited hydroperoxide interaction with this form of the enzyme. Phenolic antioxidants, only slightly extended lag periods while inhibiting oxygenation rates more than 50%. Low levels of phenol (which is normally stimulatory) or alpha-naphthol when combined with NaCN or glutathione peroxidase (agents which interfere with peroxide activation) resulted in potent synergistic inhibition with long lag times. A mechanism consistent with all of the above properties of cyclooxygenase has been elucidated, Further mechanistic explanation was sought for reaction-catalyzed self-inactivation of cyclooxygenase. This phenomenon could not be explained simply by heme lability, or cyclooxygenase sensitivity to destruction by ambient hydroperoxides, Rather, it appears to involve a destructive reaction intermediate intrinsic to involve a destructive reaction intermediate intrinsic to the cyclooxygenase mechanism.     

OH-induced free radicals in 3'-UMP and poly(U): spin-trapping and radical chromatography

Characterization of OH-induced free radicals using 3'-UMP and poly(U) was performed by a method combining spin-trapping and radical chromatography. A N2O-saturated aqueous solution containing 3'-UMP and 2-methyl-2-nitrosopropane as a spin-trap was X-irradiated. The spin adducts generated by the reactions of OH radicals with 3'-UMP were separated by paired-ion HPLC and the separated spin adducts were identified by ESR spectroscopy. In the case of poly(U), the spin adducts were digested to oligonucleotides with RNase A and then separated and identified in the same manner as 3'-UMP. The free radicals observed for poly(U) were identical to those for 3'-UMP. The 5-yl radical and the 6-yl radical were identified as precursors of various oxidized products of the base moiety, and the 4'-yl radical and 5'-yl radical, formed by H-abstraction at the C-4' and C-5' positions of the sugar moieties, respectively, were identified as precursors of strand breaks. The 1'-yl radical, produced by H-abstraction at the C-1' position of the sugar moiety, was also identified. From the similarity of the free radicals of 3'-UMP and poly(U), it is suggested that the reactivities of OH radicals with nucleotides are identical to those in polynucleotides.     

N-acyl dehydroalanines scavenge oxygen radicals and inhibit in vitro free radical mediated processes

N-substituted dehydroalanines react with and scavenge oxygen radicals. One of those compounds, the para-methoxyphenylacetyl dehydroalanine derivative, indexed as AD-5, inhibits the reduction of ferricytochrome c by superoxide anion (O2-.). It can also inhibit the oxidation of linolenic acid, another chemical process, which is mediated by hydroxyl radical (HO.). Furthermore, microsomal lipid peroxidation induced by iron salts was also inhibited by AD 5, but with a different degree of efficacy. In fact, lipid peroxidation initiated by a ferrous-oxygen complex (as in iron/NADPH-dependent peroxidation) was inhibited by AD 5 in a range of concentration of 2-4 mM. On the contrary, iron/NADPH-independent lipid peroxidation, where alkoxy radicals (RO.) have principally been involved, was inhibited in a range of concentration of 6-10 mM. The ESR studies by using the spin trapping agent DMPO, show that AD-5 reacts with HO. with a second order constant of 2.8 X 10(9)-4.5 X 10(9) M-1 s-1.     

Monoterpene biosynthesis: mechanism and stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate

The conversion of geranyl pyrophosphate to (+)-cis- and (+)-trans-sabinene hydrate by a partially purified cyclase from sweet marjoram (Majorana hortensis) is considered to proceed by the initial ionization and isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound tertiary allylic intermediate to the monocyclic (+)-(4R)-alpha-terpinyl cation. A 1,2-hydride shift and a second cyclization with water capture of the resulting cation complete the reaction sequence. [6-3H, 14C]Geranyl pyrophosphate, coupled with selective chemical degradation of the resulting sabinene hydrate products, was employed to demonstrate the hydride shift, while separate testing of the linalyl pyrophosphate enantiomers confirmed the involvement of the (3R)-antipode in the cyclization and indicated the cyclization of linalyl pyrophosphate to be faster than the coupled isomerization-cyclization of the geranyl substrate. (1R)- and (1S)-[1-3H, 14C]geranyl pyrophosphates, in conjunction with stereoselective degradations of the biosynthetic products to locate the 3H, were exploited to deduce that configuration at C1 of the substrate was retained in the reaction. These findings suggest the isomerization of the geranyl substrate to be a suprafacial process and the cyclization of the (3R)-linalyl intermediate to proceed via the anti,endo-conformation consistent with the stereo-chemistry of other monoterpene cyclizations and with chemical model studies. Sulfonium ion analogs of the presumptive linalyl and alpha-terpinyl cationic intermediates of the isomerization-cyclization sequence were shown to be potent inhibitors of the enzymatic reaction (Ki = 0.3 and 2.8 microM, respectively), and inhibition was synergized by the presence of inorganic pyrophosphate, indicating that the enzyme recognized and bound more tightly to these ion-paired species than to either cationic or anionic partner alone. Additionally, the enzyme was capable of ionizing (solvolyzing) the noncyclizable substrate analogs 6,7-dihydrogeranyl pyrophosphate and 2,3-methanogeranyl pyrophosphate. These results define the overall stereochemistry of the coupled isomerization-cyclization to sabinene hydrate, demonstrate the 1,2-hydride shift, and confirm the electrophilic nature of this enzymatic reaction type.     

Characterization of an allylic analogue of the 5'-deoxyadenosyl radical: an intermediate in the reaction of lysine 2,3-aminomutase.

An allylic analogue of the 5'-deoxyadenosyl radical has been characterized at the active site of lysine 2,3-aminomutase (LAM) by electron paramagnetic resonance (EPR) spectroscopy. The anhydroadenosyl radical, 5'-deoxy-3',4'-anhydroadenosine-5'-yl, is a surrogate of the less stable 5'-deoxyadenosyl radical, which has never been observed but has been postulated to be a radical intermediate in the catalytic cycles of a number of enzymes. An earlier communication [Magnusson, O.Th., Reed, G. H., and Frey, P. A. (1999) J. Am. Chem. Soc. 121, 9764-9765] included the initial spectroscopic identification at 77 K of the radical, which is formed upon replacement of S-adenosylmethionine by S-3',4'-anhydroadenosylmethionine as a coenzyme for LAM. The electron paramagnetic resonance spectrum of the radical changes dramatically between 77 and 4.5 K. This unusual temperature dependence is attributed to a spin-spin interaction between the radical and thermally populated, higher spin states of the [4Fe-4S]+2 center, which is diamagnetic at 4.5 K. The EPR spectra of the radical at 4.5 K have been analyzed using isotopic substitutions and simulations. Analysis of the nuclear hyperfine splitting shows that the unpaired spin is distributed equally between C5'- and C3'- as expected for an allylic radical. Hyperfine splitting from the beta-proton at C-2'(H) shows that the dihedral angle to the p(z)-orbital at C-3' is approximately 37 degrees. This conformation is in good agreement with a structural model of the radical. The rate of formation of the allylic radical shows that it is kinetically competent as an intermediate. Measurements of 2H kinetic isotope effects indicate that with lysine as the substrate, the rate-limiting steps follow initial reductive cleavage of the coenzyme analogue.     

Comparative study of the enzymatic defense systems against oxygen-derived free radicals: the key role of glutathione peroxidase

Human WI-38 diploid fibroblasts have been cultivated under high toxic O2 pressure, and their survival curves are reported. Superoxide dismutase, catalase, or glutathione peroxidase provided some protection when injected in the cells exposed to O2. This protective effect, recorded after 3 or 4 days of incubation, was the most pronounced when cells were injected just before oxygen exposure. Quantitative injection assays have been performed for the three enzymes. Surprisingly, glutathione peroxidase was found to be much more effective than both catalase and superoxide dismutase, the latter being particularly inefficient.