Peroxidase-catalyzed S-oxygenation: mechanism of oxygen transfer for lactoperoxidase

The mechanism of organosulfur oxygenation by peroxidases [lactoperoxidase (LPX), chloroperoxidase, thyroid peroxidase, and horseradish peroxidase] and hydrogen peroxide was investigated by use of para-substituted thiobenzamides and thioanisoles. The rate constants for thiobenzamide oxygenation by LPX/H2O2 were found to correlate with calculated vertical ionization potentials, suggesting rate-limiting single-electron transfer between LPX compound I and the organosulfur substrate. The incorporation of oxygen from 18O-labeled hydrogen peroxide, water, and molecular oxygen into sulfoxides during peroxidase-catalyzed S-oxygenation reactions was determined by LC- and GC-MS. All peroxidases tested catalyzed essentially quantitative oxygen transfer from 18O-labeled hydrogen peroxide into thiobenzamide S-oxide, suggesting that oxygen rebound from the oxoferryl heme is tightly coupled with the initial electron transfer in the active site. Experiments using H2(18)O2, 18O2, and H2(18)O showed that LPX catalyzed approximately 85, 22, and 0% 18O-incorporation into thioanisole sulfoxide oxygen, respectively. These results are consistent with a active site controlled mechanism in which the protein radical form of LPX compound I is an intermediate in LPX-mediated sulfoxidation reactions.     

Electrostatic modulation of electron transfer in the active site of heme peroxidases

 The heme enyzmes cytochrome c peroxidase (CCP) and pea cytosolic ascorbate peroxidase (APX) show a high level of sequence identity. The main difference near the active sites is the presence of a cation binding site in APX located about 1 nm from the Trp-179 side chain, which is hydrogen-bonded to Asp-208. It is possible that this difference in electrostatics provided by the protein environment is an essential determinant of the stabilization of the ion-pair or neutral form of the Trp...Asp couple in APX and CCP. Semiempirical molecular orbital calculations support the hypothesis that the position of the moving proton inside the couple influences the location of the free electron, leading to radical formation either on the heme or on the Trp side chain of these enzymes. Received, accepted: 26 November 1996     

Distinct heme-substrate interactions of lactoperoxidase probed by resonance Raman spectroscopy: difference between animal and plant peroxidases

Resonance Raman scattering from cow milk lactoperoxidase (LPO) and its complexes with various electron donors and inhibitors was investigated. The Raman spectrum of LPO is strikingly close to that of hog intestinal peroxidase but distinctly dissimilar to that of horseradish peroxidase (HRP). The v10 frequency suggested the six-coordinate high-spin structure of heme for native LPO in contrast with the five-coordinate high-spin structure for HRP. For the v10 band, benzohydroxamic acid caused a frequency shift with HRP but not with LPO. Guaiacol, o-toluidine, and histidine brought about a frequency shift of the v4 mode for LPO but not for HRP. The frequency shift was restored upon removal of the substrate or inhibitor by dialysis. The down shift of the v4 frequency is considered to represent an appreciable donation of electrons from the substrate or inhibitor to the porphyrin LUMO and thus their direct interaction with the heme group. From the relative intensity of the shifted and unshifted v4 lines, the dissociation constant was determined to be Kd = 52 mM for guaiacol and Kd = 87 mM for histidine at pH 7.4. The binding of histidine was relatively retarded in the presence of sulfate anion (Kd = 150 mM for 0.53 M sulfate present), and imidazole alone yielded no frequency shift, indicating the binding of the carboxyl group of histidine to the protein cationic site on one hand and a weak charge-transfer interaction between the imidazole group and the heme group on the other.     

Electron transfer within nitrogenase: evidence for a deficit-spending mechanism

The reduction of substrates catalyzed by nitrogenase utilizes an electron transfer (ET) chain comprised of three metalloclusters distributed between the two component proteins, designated as the Fe protein and the MoFe protein. The flow of electrons through these three metalloclusters involves ET from the [4Fe-4S] cluster located within the Fe protein to an [8Fe-7S] cluster, called the P cluster, located within the MoFe protein and ET from the P cluster to the active site [7Fe-9S-X-Mo-homocitrate] cluster called FeMo-cofactor, also located within the MoFe protein. The order of these two electron transfer events, the relevant oxidation states of the P-cluster, and the role(s) of ATP, which is obligatory for ET, remain unknown. In the present work, the electron transfer process was examined by stopped-flow spectrophotometry using the wild-type MoFe protein and two variant MoFe proteins, one having the β-188(Ser) residue substituted by cysteine and the other having the β-153(Cys) residue deleted. The data support a "deficit-spending" model of electron transfer where the first event (rate constant 168 s(-1)) is ET from the P cluster to FeMo-cofactor and the second, "backfill", event is fast ET (rate constant >1700 s(-1)) from the Fe protein [4Fe-4S] cluster to the oxidized P cluster. Changes in osmotic pressure reveal that the first electron transfer is conformationally gated, whereas the second is not. The data for the β-153(Cys) deletion MoFe protein variant provide an argument against an alternative two-step "hopping" ET model that reverses the two ET steps, with the Fe protein first transferring an electron to the P cluster, which in turn transfers an electron to FeMo-cofactor. The roles for ATP binding and hydrolysis in controlling the ET reactions were examined using βγ-methylene-ATP as a prehydrolysis ATP analogue and ADP + AlF(4)(-) as a posthydrolysis analogue (a mimic of ADP + P(i)).     

The cytotoxic activity of lactoperoxidase: enhancement and inhibition by neuroactive compounds

Neuronal death associated with Parkinson's disease is commonly believed to be caused by oxygen- and nitrogen-derived free radical species. Some years ago, however, we showed that peroxidase from the midbrain of dogs is able to kill various cell types, including neuroblastoma cells (M. B. Grisham et al., J. Neurochem. 48: 876-882: 1987). We postulated that a nigral peroxidase may play a significant role in the degeneration of dopaminergic neurons in Parkinson's disease. To further establish proof of principle, we recently performed a series of experiments using horseradish peroxidase and lactoperoxidase. We showed that the cytotoxic activity of lactoperoxidase is fully inhibited by physiological concentrations of dopamine, reduced glutathione, and L-cysteine, as well as by micromolar concentrations of apomorphine, desferal, aspirin, and uric acid. l-Methyl-4-phenyl-1,2-dihydropyridine (MPDP) and l-methyl-4-phenylpyridinium (MPP+) augment the cytotoxic activity, whereas l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, deprenyl, and pargyline had minimal or no effect. We also showed that horseradish peroxidase catalyzes the oxidation of MPDP to MPP+. Thus, contrary to the generally accepted theory that the in vivo oxidation of MPDP occurs spontaneously, this reaction may be catalyzed by a brain peroxidase. These observations lend further support to the suggestion that a brain peroxidase may play an important role in the metabolic events associated with Parkinson's disease.     

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 one-electron oxidation of porphyrins to porphyrin pi-cation radicals by peroxidases: an electron spin resonance investigation

For the first time, the enzymatic one-electron oxidation of several naturally occurring and synthetic water-soluble porphyrins by peroxidases was investigated by ESR and optical spectroscopy. The ESR spectra of the free radical metabolites of the porphyrins were singlets (g = 2.0024, delta H = 2-3 G), which we assigned to their respective porphyrin pi-cation free radicals. Several porphyrins were investigated and ranked by the intensity of their ESR spectra (coproporphyrin III greater than coproporphyrin I greater than deuteroporphyrin IX greater than mesoporphyrin IX greater than Photofrin II greater than protoporphyrin IX greater than uroporphyrin I greater than uroporphyrin III greater than hematoporphyrin IX). The porphyrins were oxidized by several peroxidases (horseradish peroxidase, lactoperoxidase, and myeloperoxidase), yielding the same type of ESR spectra. From these results, we conclude that porphyrins are substrates for peroxidases. The changes in the visible absorbance spectra of the porphyrins during enzymatic oxidation were monitored. The two-electron oxidation product, which was assigned to the dihydroxyporphyrin, was detected as an intermediate of the oxidation process. The optical spectrum of the porphyrin pi-cation free radical was not detected, probably due to its low steady-state concentration.     

Fe-haem bound to Escherichia coli bacterioferritin accelerates iron core formation by an electron transfer mechanism

BFR (bacterioferritin) is an iron storage and detoxification protein that differs from other ferritins by its ability to bind haem cofactors. Haem bound to BFR is believed to be involved in iron release and was previously thought not to play a role in iron core formation. Investigation of the effect of bound haem on formation of the iron core has been enabled in the present work by development of a method for reconstitution of BFR from Escherichia coli with exogenously added haem at elevated temperature in the presence of a relatively high concentration of sodium chloride. Kinetic analysis of iron oxidation by E. coli BFR preparations containing various amounts of haem revealed that haem bound to BFR decreases the rate of iron oxidation at the dinuclear iron ferroxidase sites but increases the rate of iron core formation. Similar kinetic analysis of BFR reconstituted with cobalt-haem revealed that this haem derivative has no influence on the rate of iron core formation. These observations argue that haem bound to E. coli BFR accelerates iron core formation by an electron-transfer-based mechanism.     

A proposed mechanism for light emission by bacterial luciferase involving dissociative electron transfer

A new mechanism that involves dissociative electron transfer in the energy transducing step is set forward for bacterial luciferase catalyzed light emission. The proposal involves (1) dissociation of the 4a-hydroperoxyflavin to a flavin radical and ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}$, accounting for 570 and 620nm absorption, (2) ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}$ addition to the aldehyde carbonyl to form a peroxyl radical, (3) abstraction of H from an enzyme thiol group to form RCH(OOH)OH, (4) thiyl radical abstraction of the H on C in RCH(OOH)OH, a step which can show a $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$ of ca. 4, and (5) dissociative electron- transfer, a highly exothermic step that leads to a protonated flavin excited state, a carboxylic acid and water.     

Structure and electron transfer mechanism of pyruvate:ferredoxin oxidoreductase

The first crystal structure of pyruvate:ferredoxin oxidoreductase to be determined has provided significant new information on its structural organization and redox chemistry. Spectroscopic analyses of a radical reaction intermediate have shed more light on its thiamin-based mechanism of catalysis. Different approaches have been used to study the interaction between the enzyme and ferredoxin, its redox partner.     

Selective oxygen transfer catalysed by heme peroxidases: synthetic and mechanistic aspects

The synthetic and mechanistic aspects of the use of heme peroxidases as functional mimics of the cytochrome P450 monooxygenases in oxygen-transfer reactions have been described. The chloroperoxidase from Caldariomyces fumago (CPO) is the catalyst of choice in sulfoxidation, hydroxylation and epoxidation on account of its high activity and enantioselectivity. Other heme peroxidases were less active by orders of magnitude; protein engineering has resulted in impressive improvements but even the most active mutant was still at least an order of magnitude less active than CPO. The 'oxygen-rebound' mechanisms of oxygen transfer mediated by heme enzymes - as originally conceived - have proved to be untenable. Dual pathway mechanisms, via oxoferryl species that insert oxygen as well as iron hydroperoxide species that insert OH(+), have been proposed that accommodate all of the known experimental data.     

Intramolecular electron transfer versus substrate oxidation in lactoperoxidase: investigation of radical intermediates by stopped-flow absorption spectrophotometry and (9-285 GHz) electron paramagnetic resonance spectroscopy

We have combined the information obtained from rapid-scan electronic absorption spectrophotometry and multifrequency (9-295 GHz) electron paramagnetic resonance (EPR) spectroscopy to unequivocally determine the electronic nature of the intermediates in milk lactoperoxidase as a function of pH and to monitor their reactivity with organic substrates selected by their different accessibilities to the heme site. The aim was to address the question of the putative catalytic role of the protein-based radicals. This experimental approach allowed us to discriminate between the protein-based radical intermediates and [Fe(IV)=O] species, as well as to directly detect the oxidation products by EPR. The advantageous resolution of the g anisotropy of the Tyr (*) EPR spectrum at high fields showed that the tyrosine of the [Fe(IV)=O Tyr (*)] intermediate has an electropositive and pH-dependent microenvironment [g(x) value of 2.0077(0) at pH >or= 8.0 and 2.0066(2) at 4.0 相似文献   

Oxygenation of monounsaturated fatty acids by soybean lipoxygenase-1: evidence for transient hydroperoxide formation

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Previous work from our laboratories has demonstrated that SBLO-1 will also catalyze the oxygenation of monounsaturated acids (Clapp, C. H., Senchak, S. E., Stover, T. J., Potter, T. C., Findeis, P. M., and Novak, M. J. (2001) Soybean Lipoxygenase-Mediated Oxygenation of Monounsaturated Fatty Acids to Enones, J. Am. Chem. Soc. 123, 747-748). Interestingly, the products are alpha,beta-unsaturated ketones rather than the expected allylic hydroperoxides. In the present work, we provide evidence that the monoolefin substrates are initially converted to allylic hydroperoxides, which are subsequently converted to the enone products. The hydroperoxide intermediates can be trapped by reduction to the corresponding allylic alcohols with glutathione peroxidase plus glutathione or with SnCl2. Under some conditions, the hydroperoxide intermediates accumulate and can be detected by HPLC and peroxide assays. Kinetics measurements at low concentrations of [1-14C]-9(Z)-octadecenoic acid indicate that oxygenation of this substrate at 25 degrees C, pH 9.0 occurs with kcat/Km = 1.6 (+/-0.1) x 10(2) M-1 s-1, which is about 105 lower than kcat/Km for oxygenation of 9(Z),12(Z)-octadecadienoic acid (linoleic acid). Comparison of the activities of 9(Z)-octadecenoic acid and 12(Z)-octadecenoic acid implies that the two double bonds of linoleic acid contribute almost equally to the C-H bond-breaking step in the normal lipoxygenase reaction. The results are consistent with the notion that SBLO-1 functionalizes substrates by a radical mechanism.     

NBU1 integrase: evidence for an altered recombination mechanism

NBU1 is a 10.3 kbp Bacteroides mobilizable transposon. A previous study had identified a 2.7 kbp segment of the excised circular intermediate that was sufficient to mediate integration of the element after transfer. This segment contained an integrase gene, intN1, and a region spanning the ends of the circular form within which integration occurred (attN1). The integrase protein, IntN1, appeared to be a member of the tyrosine recombinase family because it contains the canonical C-terminal RKHRHY [RK(H/K)R(H/W)Y] motif that characterizes members of that family. In this study, we describe an Escherichia coli-based integration assay system that has allowed us to characterize attN1 in detail. We first localized attN1 to a 250 bp region. We then used site-directed mutations to identify directly repeated sequences within attN1 that were required for site-specific integration. The locus of NBU1 site-specific integration in the Bacteroides thetaiotaomicron chromosome, attBT1-1, contains a 14 bp sequence that is identical to a 14 bp sequence that spans the joined ends of the NBU1 attN1 site (common core sequences). The effects of mutations in the common core were different from the expected results if NBU1 integration was similar to lambda integration. In particular single base changes near one end of the common core region, which introduced heterology, actually increased the frequency of integration. By contrast, compensating changes that restored homology in the common core region reduced the integration frequency. The recombination mechanism also differs from the one used by conjugative transposons that have coupling sequences between the sites of strand cleavage and exchange. These results indicate that although NBU1 integrase is considered to be a member of the tyrosine recombinase family, it catalyses an integrative recombination reaction that occurs by a different crossover mechanism.     

Cobalt substitution supports an inner-sphere electron transfer mechanism for oxygen reduction in pea seedling amine oxidase

Copper amine oxidases (CAOs) are a large family of proteins that use molecular oxygen to oxidize amines to aldehydes with the concomitant production of hydrogen peroxide and ammonia. CAOs utilize two cofactors for this reaction: topaquinone (TPQ) and a Cu(II) ion. Two mechanisms for oxygen reduction have been proposed for these enzymes. In one mechanism (involving inner-sphere electron transfer to O₂), Cu(II) is reduced by TPQ, forming Cu(I), to which O₂ binds, forming a copper–superoxide complex. In an alternative mechanism (involving outer-sphere electron transfer to O₂), O₂ is directly reduced by TPQ, without reduction of Cu(II). Substitution of Cu(II) with Co(II) has been used to distinguish between the two mechanisms in several CAOs. Because it is unlikely that Co(II) could be reduced to Co(I) in this environment, an inner-sphere mechanism, as described above, is prevented. We adapted metal replacement methods used for other CAOs to the amine oxidase from pea seedlings (PSAO). Cobalt-substituted PSAO (CoPSAO) displayed nominal catalytic activity: k _cat is 4.7% of the native k _cat, and K _M (O₂) for CoPSAO is substantially (22-fold) higher. The greatly reduced turnover number for CoPSAO suggests that PSAO uses the inner-sphere mechanism, as has been predicted from ¹⁸O isotope effect studies (Mukherjee et al. in J Am Chem Soc 130:9459–9473, 2008), and is catalytically compromised when constrained to operate via outer-sphere electron transfer to O₂. This study, together with previous work, provides strong evidence that CAOs use both proposed mechanisms, but each homolog may prefer one mechanism over the other.     

Studies on the mechanism of the iodination of tyrosine by lactoperoxidase

Studies with lactoperoxidase showed that a highly reactive intermediate is produced (on the enzyme) from I- and H2O2 which then diffuses from the enzyme and very rapidly and indiscriminately iodinates any Tyr or peptides containing Tyr which are in the same solution. The evidence supporting these conclusions follows. 1) The rate followed the Michaelis-Menten pattern with I- and H2O2 while the concentration of Tyr peptides had no measurable effect on the rate; 2) the rates of reaction were independent of the type of peptide in which Tyr was located; 3) the amount of iodination which had occurred after the reaction had gone to completion and the amounts of monoiodination and diiodination after completion of the reaction were independent of the peptide type, the pH, the solvent polarity, or the ionic strength; 4) competition for reaction by two very different Tyr peptides depended only on their initial concentrations; and 5) iodination of a large protein occurred through a dialysis membrane. Free Tyr was iodinated at the same rate as Tyr peptides by lactoperoxidase, but monoiodotyrosine and m-fluorotyrosine were iodinated at one-half that rate. The results also showed that one can choose ratios of [peptide] to [H2O2] such that monoiodination is maximized relative to diiodination. It was also found that the iodination capacity of a mixture of I- and H2O2 with lactoperoxidase (when Tyr was absent) was only slowly dissipated. Finally, the results showed that lactoperoxidase can be used to brominate and chlorinate Tyr peptides at a slow rate.     

Oxidation of an antitumor drug ellipticine by peroxidases

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Since we found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts, this anticancer drug is considered to function as a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its enzymatic activation in target tissues. Here, we demonstrate that ellipticine is also oxidized by peroxidases, which are abundantly expressed in several target tumor tissues. Lactoperoxidase, myeloperoxidase and horseradish peroxidase were used as models. Peroxidases in the presence of hydrogen peroxide oxidize ellipticine to an ellipticine dimer and N(2)-oxide of ellipticine as the major and minor metabolite, respectively. Inhibition of the peroxidase-mediated ellipticine oxidation by radical scavengers ascorbate, glutathione and NADH suggests a one-electron mechanism of the oxidation. The implication of the oxidation of ellipticine by peroxidases in its mechanism of action is discussed.