Ellagitannin biosynthesis: laccase-catalyzed dimerization of tellimagrandin II to cornusiin E in Tellima grandiflora

An enzyme has been purified from leaves of the weed Tellima grandiflora (fringe cups, Saxifragaceae) that catalyzed the O2-dependent oxidation of the monomeric ellagitannin, tellimagrandin II, to a dimeric derivative, cornusiin E. The apparently homogeneous enzyme preparation had a Mr of ca. 160,000 (with four subunits of Mr 40,000), a pH-optimum and an isoelectric point at pH 5.2, and was most stable at pH 4.3. Inhibition studies revealed that this new enzyme, for which the systematic name 'tellimagrandin II: O2 oxidoreductase' is proposed, is a member of the laccase (EC 1.10.3.2) family of phenol oxidases. The properties of this enzyme differed from that of a related laccase that catalyzed the transition of 1,2,3,4,6-pentagalloylglucopyranose to tellimagrandin II, the preceding step in the biosynthetic route to cornusin E.     

Biosynthesis of the dimeric ellagitannin,cornusiin E,in Tellima grandiflora

First evidence for the in vitro synthesis of a dimeric ellagitannin has been obtained with cell-free extracts from the weed Tellima grandiflora (fringe cups, Saxifragaceae). Partially purified enzyme preparations from leaves of this plant catalyzed the oxidation of 1,2,3,4,6-pentagalloyl-beta-D-glucose to the monomeric ellagitannin, tellimagrandin II, followed by oxidative coupling of two units of this intermediate to yield a dimeric derivative. Chemical degradation, MALDI-TOF mass spectrometry, 1H and 13C nuclear magnetic resonance, and CD spectroscopy were employed to identify this enzyme reaction product as cornusiin E which is characterized by a (S)-valoneoyl bridge between glucose-positions 2, 4' and 6'. This result was supported by comparison with data obtained for cornusiin E that had been isolated from leaves of intact T. grandiflora plants. No indication for the earlier proposed existence of rugosin D (an isomer with a 1,4',6'-bound valoneoyl unit) in T. grandiflora has been obtained in this investigation.     

Coordination structures and reactivities of compound II in iron and manganese horseradish peroxidases. A resonance Raman study

Resonance Raman investigations on compound II of native, diacetyldeuteroheme-, and manganese-substituted horseradish peroxidase (isozyme C) revealed that the metal-oxygen linkage in the compound differed from one another in its bond strength and/or structure. Fe(IV) = O stretching frequency for compound II of native enzyme was pH sensitive, giving the Raman line at 772 and 789 cm-1 at pH 7 and 10, respectively. The results confirmed the presence of a hydrogen bond between the oxo-ligand and a nearby amino acid residue (Sitter, A. J., Reczek, C. M., and Terner, J. (1985) J. Biol. Chem. 260, 7515-7522). The Fe(IV) = O stretch for compound II of diacetylheme-enzyme was located at 781 cm-1 at pH 7 which was 9 cm-1 higher than that of native enzyme compound II. At pH 10, however, the Fe(IV) = O stretch was found at 790 cm-1, essentially the same frequency as that of native enzyme compound II. The pK value for the pH transition, 8.5, was also the same as that of native compound II. Unlike in native enzyme, D2O-H2O exchange did not cause a shift of the Fe(IV) = O frequency of diacetylheme-enzyme. Thus, the metal-oxygen bond at pH 7 was stronger in diacetylheme-enzyme due to a weaker hydrogen bonding to the oxo-ligand, while the Fe(IV) = O bond strength became essentially the same between both enzymes at alkaline pH upon disruption of the hydrogen bond. A much lower reactivity of the diacetylheme-enzyme compound II was accounted to be due to the weaker hydrogen bond. Compound II of manganese-substituted enzyme exhibited Mn(IV)-oxygen stretch about 630 cm-1, which was pH insensitive but down-shifted by 18 cm-1 upon the D2O-H2O exchange. The finding indicates that its structure is in Mn(IV)-OH, where the proton is exchangeable with a water proton. These results establish that the structure of native enzyme compound II is Fe(IV) = O but not Fe(IV)-OH.     

Characterization of arylsulfatase activity in brine shrimp, Artemia salina

Arylsulfatase from Artemia salina exists in at least two forms (AS I and AS II). The paper presents characterization of the AS II form of the arylsulfatase. The enzyme was able to hydrolyze p-nitrocatechol sulfate (pNCS) as well as ascorbate sulfate. It exhibited maximum activity at temperature of 50 °C and was stable for 2 h at 4-10 °C. Optimum pH shifted from 6.2 at 4 mM pNCS (substrate) to 4.8 at 20 mM pNCS. The enzyme displayed linear kinetics. AS II arylsulfatase exists in two molecular forms (349 and 460 kDa) composed of identical subunits with molecular mass of 53 kDa. Sulfite and phosphate ions were the most potent inhibitors of the enzyme. Cyanide proved to be a weak inhibitor. Sulfate and low concentrations of silver ions had no effect on the enzyme activity. Based on the above results, modifications in the assay for determination of enzyme activity are proposed.     

Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O

We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.     

Gut pH, redox conditions and oxygen levels in an aquatic caterpillar: potential effects on the fate of ingested tannins

Larvae of the freshwater moth Acentria ephemerella (Pyralidae, Lepidoptera) can fully develop on Myriophyllum spicatum, a submerged macrophyte containing 7-10% of its dry mass as tannins. We investigated the physicochemical gut parameters of larvae fed with M. spicatum or Potamogeton perfoliatus, a food plant lacking tannins, and the chemical fate of ingested polyphenols. Microelectrode studies revealed that larval midguts were slightly alkaline (pH 8) and had a positive redox potential. Whole guts were oxygen sinks owing to the oxygen demand of the gut contents. Oxygen penetrated the midgut up to 100 microm, but the centres of the foregut and midgut were always anoxic. The physicochemical parameters of the guts did not change with the food plant. The major tannin from M. spicatum, tellimagrandin II, was significantly depleted in the midgut and was not detected in faeces. In vitro studies indicated that tellimagrandin II is rapidly depleted mainly through oxidation, and hydrolysis might also occur. Our findings for A. ephemerella are compared with those for terrestrial Lepidoptera, and possible mechanisms for adaptations to tannin-rich food plants are discussed.     

Effects of N-alkyl and ammonium groups on the hydrolytic cleavage of DNA with a Cu(II)TACH (1,3,5-triaminocyclohexane) complex. Speciation, kinetic, and DNA-binding studies for reaction mechanism

cis,cis-1,3,5-Triaminocyclohexane (c-TACH), its N-alkyl-derivatives (alkyl = methyl, ethyl), and trans,cis-1,3,5-triaminocyclohexane (t-TACH) were prepared, and speciation and DNA cleaving property of Cu(II) complexes of these ligands were investigated. All of the complexes efficiently promote the hydrolytic cleavage of supercoiled plasmid DNA under physiological conditions without further additives. The DNA cleavage rate (V(obs)) trend at pH values between 8 and 9 is N-Me(3) = N-Et(1) < t-TACH < c-TACH < N-Et(2) < N-Et(3). At pH 7, the trend is c-TACH < N-Et(3) = N-Et(2) < N-Et(1) < N-Me(3) < t-TACH. The cleavage rate constants at 35 degrees C, for the c-TACH complex are 3 x 10(-1) h(-1) at pH 8.1 and 2 x 10(-1) h(-1) at pH 7.0 ([DNA] = 7 microM, [Cu(II)-complex] = 105 microM). The hydrolytically active species at pH > 8 is CuL(H(2)O)(OH)(+) in which L coordinates to Cu(II) as a tridentate ligand for all complexes except for t-TACH. The hydrolytically active species at pH 7 is CuLH(H(2)O)(3)(3+) or CuLH(H(2)O)(4)(3+) in which LH coordinates as bidentate ligand. DNA-binding constants of c-TACH and t-TACH complexes are presented and the effects of N-alkyl and ammonium groups are discussed in light of the proposed reaction mechanism.     

Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.     

Mn(II) oxidation is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium

The manganese peroxidase (MnP), from the lignin-degrading fungus Phanerochaete chrysosporium, an H2O2-dependent heme enzyme, oxidizes a variety of organic compounds but only in the presence of Mn(II). The homogeneous enzyme rapidly oxidizes Mn(II) to Mn(III) with a pH optimum of 5.0; the latter was detected by the characteristic spectrum of its lactate complex. In the presence of H2O2 the enzyme oxidizes Mn(II) significantly faster than it oxidizes all other substrates. Addition of 1 M equivalent of H2O2 to the native enzyme in 20 mM Na-succinate, pH 4.5, yields MnP compound II, characterized by a Soret maximum at 416 nm. Subsequent addition of 1 M equivalent of Mn(II) to the compound II form of the enzyme results in its rapid reduction to the native Fe3+ species. Mn(III)-lactate oxidizes all of the compounds which are oxidized by the enzymatic system. The relative rates of oxidation of various substrates by the enzymatic and chemical systems are similar. In addition, when separated from the polymeric dye Poly B by a semipermeable membrane, the enzyme in the presence of Mn(II)-lactate and H2O2 oxidizes the substrate. All of these results indicate that the enzyme oxidizes Mn(II) to Mn(III) and that the Mn(III) complexed to lactate or other alpha-hydroxy acids acts as an obligatory oxidation intermediate in the oxidation of various dyes and lignin model compounds. In the absence of exogenous H2O2, the Mn-peroxidase oxidized NADH to NAD+, generating H2O2 in the process. The H2O2 generated by the oxidation of NADH could be utilized by the enzyme to oxidize a variety of other substrates.     

Petunia peroxidase a: isolation, purification and characteristics

The fast-moving anionic peroxidase isoenzyme variant PRXa was purified from leaves of petunia (Petunia hybrida). Over 1300-fold purification was achieved by subjecting extracellular extracts to two sequential acetone precipitations and resuspending the pellets at pH 5.0 and pH 8.0, respectively, followed by gel filtration and chromatofocusing. The purified enzyme had an absorbance ratio (A405 nm/A280 nm) of 3.6, a molecular mass of about 37 kDa and a pI of 3.8. Three molecular forms with slightly different molecular masses were separated by concanavalin-A--Sepharose affinity chromatography, indicating that these three forms differ in their carbohydrate moieties. The absorption spectrum of PRXa had maxima at 496 and 636 nm and a Soret band at 405 nm. Spectra of compounds I and IV were obtained by titrating a batch of PRXa stored for several months at -20 degrees C with H2O2. The addition of 1 mol H2O2/mol freshly purified PRXa caused the formation of compound II, indicating that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. Compound III was obtained from both preparations in the presence of excess H2O2. The pH optimum of PRXa for the reaction with H2O2 and guaiacol was 5.0 and its specific activity 61 mkat/g protein. Among various aromatic compounds, coniferyl alcohol was polymerized by PRXa to presumed lignin-like material. The extracellular localization and high affinity of PRXa for the cinnamic acid derivatives suggest that this isoenzyme functions in the polymerization or cross-linking of lignin in the plant cell wall.     

Polyphenolic allelochemicals from the aquatic angiosperm Myriophyllum spicatum inhibit photosystem II

Myriophyllum spicatum (Haloragaceae) is a highly competitive freshwater macrophyte that produces and releases algicidal and cyanobactericidal polyphenols. Among them, beta-1,2,3-tri-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-D-glucose (tellimagrandin II) is the major active substance and is an effective inhibitor of microalgal exoenzymes. However, this mode of action does not fully explain the strong allelopathic activity observed in bioassays. Lipophilic extracts of M. spicatum inhibit photosynthetic oxygen evolution of intact cyanobacteria and other photoautotrophs. Fractionation of the extract provided evidence for tellimagrandin II as the active compound. Separate measurements of photosystem I and II activity with spinach (Spinacia oleracea) thylakoid membranes indicated that the site of inhibition is located at photosystem II (PSII). In thermoluminescence measurements with thylakoid membranes and PSII-enriched membrane fragments M. spicatum extracts shifted the maximum temperature of the B-band (S(2)Q(B)(-) recombination) to higher temperatures. Purified tellimagrandin II in concentrations as low as 3 microM caused a comparable shift of the B-band. This demonstrates that the target site of this inhibitor is different from the Q(B)-binding site, a common target of commercial herbicides like 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Measurements with electron paramagnetic resonance spectroscopy suggest a higher redox midpoint potential for the non-heme iron, located between the primary and the secondary quinone electron acceptors, Q(A) and Q(B). Thus, tellimagrandin II has at least two modes of action, inhibition of exoenzymes and inhibition of PSII. Multiple target sites are a common characteristic of many potent allelochemicals.     

Inhibition of lignin peroxidase H2 by sodium azide.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 from Phanerochaete chrysosporium and H2O2 was strongly inhibited by sodium azide. Inhibition was competitive with respect to veratryl alcohol (Ki = 1-2 microM) and uncompetitive with respect to H2O2. In contrast, sodium azide bound to the native enzyme at pH 6.0 with an apparent dissociation constant (KD) of 126 mM. Formation of azidyl radicals was detected by ESR spin trapping techniques. The enzymes is nearly completely inactivated in four turnovers. The H2O2-activated enzyme intermediate (compound I) reacted with sodium azide to form a new species rather than be reduced to the enzyme intermediate compound II. The new species has absorption maxima at 418, 540, and 570 nm, suggesting the formation of a ferrous-lignin peroxidase-NO complex. Confirmation of this assignment was obtained by low-temperature ESR spectroscopy. An identical complex could be simulated by the addition of nitrite to the reduced enzyme. The enzyme intermediate compound II is readily reduced by sodium azide to native enzyme with essentially no loss of activity.     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The K(m) values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 microM, respectively. The calculated k(cat) value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25 degrees C was 6.7s(-1), giving a k(cat)/K(m) value of 0.32 microM(-1)s(-1). The k(cat) value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25 degrees C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.     

The chlorinating activity of human myeloperoxidase: high initial activity at neutral pH value and activation by electron donors

The steady-state activity of myeloperoxidase in the chlorination of monochlorodimedone at neutral pH was investigated. Using a stopped-flow spectrophotometer we were able to show that the enzymic activity at pH 7.2 rapidly declined in time. During the first 50-100 ms after addition of H2O2 to the enzyme, a turnover number of about 320 s-1 per haem was observed. However, this activity decreased rapidly to a value of about 25s-1 after 1 s. This shows that in classical steady-state activity measurements, the real activity of the enzyme at neutral pH is grossly underestimated. By following the transient spectra of myeloperoxidase during turnover it was shown that the decrease in activity was probably caused by the formation of an enzymically inactive form of the enzyme, Compound II. As demonstrated before (Bolscher, B.G.J.M., Zoutberg, G.R., Cuperus, R.A. and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191) reductants such as ascorbic acid and ferrocyanide convert Compound II, which accumulates during turnover, into active myeloperoxidase. Activity measurements in the presence of ascorbic acid showed, indeed, that the moderate enzymic activity was higher than in the absence of ascorbic acid. With 5-aminosalicylic acid present, however, the myeloperoxidase activity remained at a much higher level, namely about 150 s-1 per haem during the time interval from 100 ms to 5 s after mixing. From combined stopped-flow/rapid-scan experiments during turnover it became clear that in the presence of 5-aminosalicylic acid the initially formed Compound II was rapidly converted back to native enzyme. Presteady-state experiments showed that 5-aminosalicylic acid reacted with Compound II with a K2 of 3.2 x 10(5) M-1.s-1, whereas for ascorbic acid a K2 of 1.5 x 10(4) M-1.s-1 was measured at pH 7.2. In the presence of 5-aminosalicylic acid during the time interval in which the myeloperoxidase activity remained constant, a Km for H2O2 at pH 7.2 was determined of about 30 microM at 200 mM chloride. In the absence of reductants the same value was found during the first 100 ms after addition of H2O2 to the enzyme. The physiological consequences of these findings are discussed.     

Enzymology of gallotannin and ellagitannin biosynthesis

Gallotannins and ellagitannins, the two subclasses of hydrolyzable tannins, are derivatives of 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose. Enzyme studies with extracts from oak leaves (Quercus robur, syn. Quercus pedunculata; Quercus rubra) and from staghorn sumac (Rhus typhina) revealed that this pivotal intermediate is synthesized from beta-glucogallin (1-O-galloyl-beta-D-glucopyranose) by a series of strictly position-specific galloylation steps, affording so-called 'simple' gallotannins, i.e., mono- to pentagallyoylglucose esters. Besides its role as starter molecule, beta-glucogallin was also recognized as the principal energy-rich acyl donor required in these transformations. Subsequent pathways to 'complex' gallotannins have recently been elucidated by the isolation of five different enzymes from sumac leaves that were purified to apparent homogeneity. They catalyzed the beta-glucogallin-dependent galloylation of pentagallyoylglucose to a variety of hexa- and heptagalloylglucoses, plus several not yet characterized higher substituted analogous galloylglucoses. With respect to the biosynthesis of ellagitannins, postulates that had been formulated already decades ago were proven by the purification of a new laccase-like phenol oxidase from leaves of fringe cups (Tellima grandiflora) that regio- and stereospecifically oxidized pentagallyoylglucose to the monomeric ellagitannin, tellimagrandin II. This compound was further oxidized by a similar but different laccase-like oxidase to yield a dimeric ellagitannin, cornusiin E.     

Steady-state and transient-state kinetic studies on the oxidation of 3,4-dimethoxybenzyl alcohol catalyzed by the ligninase of Phanerocheate chrysosporium Burds

Catalysis of the H2O2-dependent oxidation of 3,4-dimethoxybenzyl (veratryl) alcohol by the hemoprotein ligninase isolated from wood-decaying fungus, Phanerochaete chrysosporium Burds, is characterized. The reaction yields veratraldehyde and exhibits a stoichiometry of one H2O2 consumed per aldehyde formed. Ping-pong steady-state kinetics are observed for H2O2 (KM = 29 microM) and veratryl alcohol (KM = 72 microM) at pH 3.5. The magnitude of the turnover number varies from 2 to 3 s-1 at this pH, depending on the preparation of the enzyme. Each preparation of enzyme consists of a mixture of active and inactive enzyme. Extensive steady-state kinetic studies of several different preparations of enzyme, suggest a mechanism in which H2O2 reacts with enzyme to form an intermediate that subsequently reacts with the alcohol to return the enzyme to the resting state. The pH dependence of the overall reaction indicates that an ionization occurs having an apparent pK alpha approximately 3.1. The activity is, thus, nearly zero at pH 5 and increases to a maximum near pH approximately 2. However, the enzyme is unstable at this low pH. Transient-state kinetic studies reveal that, upon reaction of ligninase with H2O2, spectral changes occur in the Soret region, which, by analogy to previous studies of horseradish peroxidase, are consistent with formation of Compounds I and II. The active form of the enzyme appears to react rapidly with H2O2; we observed a positive correlation between the turnover number of the enzyme preparation and the extent of a rapid reaction between H2O2 and ligninase to form Compound I. Free radical cations derived from veratryl alcohol do not appear to be released from the enzyme during catalysis; however, other substrates are known to be converted to cation radicals (Kersten, P., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612). Our results are generally consistent with a classical peroxidase mechanism for the action of ligninase on lignin-like substrates.     

On the role of bicarbonate in peroxidations catalyzed by Cu,Zn superoxide dismutase

The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25 degrees C and pH 7.2, is not influenced by 10-mM HCO3-; whereas the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline sulfonate] (ABTS=) is virtually completely dependent upon HCO3-. The reduction of the active site Cu(II) by H2O2, which precedes inactivation of the enzyme, occurred at the same rate in phosphate buffer with or without bicarbonate added. These results indicate that HCO3- does not play a role in facilitating the interaction of H2O2 with the active site copper, but they can be accommodated by the proposal that HCO3- is oxidized to HCO3*, which then diffuses from that site and causes the oxidation of substrates, such as ABTS=, that are too large to traverse the solvent access channel to the Cu(II).     

Kinetics, anion binding and mechanism of Co(II)-substituted bovine muscle carbonic anhydrase

The binding of N3- to Co(II)-substituted bovine carbonic anhydrase III was measured at various pH values by spectrophotometric titrations. The apparent Ki values were found to increase with pH in the studied range between pH 5.8 and 8.9. The inhibition of CO2 hydration by N-3 was found to be essentially uncompetitive at all investigated pH values (pH 6.3-8.9). The Ki values for the inhibition of kcat are much smaller than those obtained in the spectrophotometric titrations indicating that an enzyme form with a high affinity for N-3, presumably having a metal-bound H2O, accumulates in the steady state at saturating CO2 concentrations. Assuming that the low pH limit of Ki = 9 microM for the inhibition of kcat represents the affinity of N-3 for the Co(II)-OH2 form, a pKa value near 5 can be estimated for Co(II)-bound water from the pH dependence of N-3 binding in the absence of CO2. Measurements of time-resolved absorption spectra during CO2 hydration in the presence of a low N-3 concentration showed the transient appearance of the characteristic spectrum of the enzyme-N-3 adduct clearly demonstrating the accumulation in the steady state of an enzyme form with a high affinity for N-3. In similar experiments without inhibitor the transient formation of a spectral form corresponding to a Co(II)-OH2 species has been demonstrated. This spectral form is rather featureless lacking the absorption maxima at 618 nm and 640 nm characteristic of the Co(II)-OH- species. Our results strongly support the hypothesis that the rate-limiting step in CO2 hydration catalyzed by carbonic anhydrase III is the protolysis of metal-bound water.     

Extended x-ray absorption fine structure and electron paramagnetic resonance of nitrous oxide reductase from Pseudomonas aeruginosa strain P2.

The copper centers of nitrous oxide reductase from Pseudomonas aeruginosa strain P2 were studied by x-ray and electron paramagnetic resonance (EPR) spectroscopy. The enzyme is dimeric and contains four Cu atoms and about seven cysteine residues/subunit of Mr = 73,000. The extended x-ray absorption fine structure (EX-AFS) spectrum was analyzed for enzyme as isolated (oxidized or slightly reduced), enzyme exposed briefly to air, reduced enzyme, and enzyme at pH 7 after having been activated by standing at pH 10. The average Cu ligand environment in the first shell was best modeled for all forms of the enzyme by a combination of N/O and S atoms at a total coordination number between 3 and 4 and bond distances ranging from 1.96-2.03 A for Cu-N/O and 2.20-2.25 A for Cu-S. The data could be fit without using Cu-Cu interactions. Overall the results are similar to those reported for the enzyme for Pseudomonas stutzeri (Scott, R. A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086). The first derivative EPR spectra of the Cu(II) centers at 15 and 45 K were qualitatively similar among enzyme as isolated and enzyme exposed to N2O or air. These three nominally oxidized samples showed an axial signal with g perpendicular = 2.03 and g parallel = 2.15-2.16. Hyperfine structure was observed in both the g parallel and g perpendicular regions with splittings of 43 and 25 gauss, respectively. These hyperfine components are attributed to exchange coupled Cu(I)-Cu(II) S = 1/2 (half-met) centers. In the enzyme as isolated and after exposure to N2O, about 3/4 of the Cu was EPR silent, whereas after exposure to air the signal integrated to about half the Cu concentration. The EPR spectrum of enzyme activated at pH 10 but frozen at pH 7 was a composite of spectra from activated and inactive species. The activated species presented a complex set of narrow hyperfine components which may arise from contributions from more than one species of half-met center.